Chemical Visualization of Phosphoproteomes on Membrane — Volkswagen Polo Vivo

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Chemical Visualization of Phosphoproteomes on

Abstract

With new discoveries of roles of phosphorylation on a daily phospho-specific antibodies, as the primary for on-membrane detection of phosphoproteins, enormous challenges. To address an need for convenient and reliable of phosphorylation events, we report a strategy for sensitive phosphorylation in the Western blotting format. The reagent, which we termed is based on a multifunctionalized soluble and is capable of selectively binding to residues independent of amino microenvironment, thus offering promise as a universal tool in analyses where the site of is not known or its specific antibody is not The specificity and sensitivity of the approach was examined using a mixture of proteins. The method was then to monitor phosphorylation changes in in kinase and phosphatase assays. to demonstrate the unique ability of to measure endogenous phosphorylation, we it to visualize and determine the differences in proteins that interact wild-type and kinase … of Polo-like kinase 1 during the results of which were confirmed by a quantitative phosphoproteomics

Protein phosphorylation is an essential modification that regulates cellular functions, including cycle progression, proliferation, signal transduction, and apoptosis (1 ). It is the most common covalent of proteins, and irregularities in phosphorylation are a major cause of onset and of many diseases, most … (2 ). Consequently, detection of phosphorylation is essential in further of the signaling pathways of an organism to and treat such abnormalities.

detection on membrane, i.e. . blotting, has remained as the most biochemical method for the analysis of including phosphorylated proteins. In a Western blotting procedure, proteins are separated on a gel, onto a membrane, and detected by or the like, a phospho-specific antibody is required to visualize specific There are general phospho-specific available, but at present, only antibodies are of the required specificity (3 ). and phosphothreonine residue detection is sequence-dependent, resulting in partiality of antibodies. Most research currently use phosphosite-specific antibodies for their protein of interest. prior knowledge of specific sites is required, thus the analysis to only well phosphorylation events. Phospho-specific typically cost much to generate, whereas the quality is difficult to control compared a general antibody.

Another used technique for phosphoprotein is radioactive labeling with 32 P (4 ). In to working with dangerous material, a major disadvantage of approach is the difficulty of identifying phosphorylation. This arises the toxic effects 32 P has on cells, DNA fragmentation, changing cell causing cell cycle and eventually resulting in apoptosis (5. 6 ). As a radioactive analyses of protein are more commonly carried out in .

Mass spectrometry has emerged as a tool to analyze protein including the identification of phosphorylation (7. 8 ), but many challenges remain because of low ionization efficiency and fragmentation of phosphopeptides (9 ). Furthermore, groups do not have access to an instrument on daily basis of high cost and technical to operate the instrument. Phosphorylation by mass spectrometry typically high resolution and high accuracy. Few bench-type instruments, are available for phosphorylation analysis, it as a routine analytical technique in a biology laboratory.

Development of a method that selectively only phosphorylated proteins the prejudice of amino acid would therefore be of high There have been a of attempts to introduce such a that could specifically and bind to a phosphate group for One of the first developments was “Stains-All,” a dye that could bind to proteins in a gel. Unfortunately, its sensitivity and a high affinity for RNA, and acidic proteins limited its applications (10. 11 ). Diamond is another phosphoprotein-specific developed by Molecular Probes. had an improved selectivity compared “Stains-All” (12. 13 ). Pro-Q dye is on fluorescent signal detection and has successfully used by some for the in-gel staining, particularly for gels (14 ⇓ –16 ). Although there attempts to utilize it on a PVDF or membrane (17 ), some background of the nonspecific binding of the reagent and the selectivity of the Pro-Q Diamond make it difficult to confidently phosphoproteins after transfer. As a Pro-Q Diamond has most been used for in-gel detection. A different staining termed Phos-tag employs Zn 2+ metal ion complex (18 ) for phosphoprotein Combined with a chemiluminescence-based system, the technique was introduced to an on PVDF membranes, enabling blot-like detection of the phosphoproteins. the Phos-tag reagent has been to detect phospho-containing species (18 ), the and non-phospho-specific signals still high (19 ). Because of these the reagent, therefore, has been used to identify phosphorylation by a gel or to detect isomers of multiphosphorylated by incorporating the reagent during the of SDS-PAGE gels (20. 21 ).

Titanium ion (IV) and TiO 2 have demonstrated to possess superior toward phosphorylated residues on occasions, particularly when “enhancers” are used (22 ⇓ –24 ). In our lab, we demonstrated the unparalleled utility of enrichment under homogeneous We have previously functionalized nanopolymer (dendrimer) with Ti(IV) ions (termed for phosphopeptide enrichment prior to spectrometry analysis, exhibiting high selectivity, sensitivity, and (25 ). Recently, we have also the successful concept of titanium-functionalized to phosphoprotein detection in ELISA which we termed pIMAGO 1 (26 ). We have demonstrated the great of the pIMAGO reagent for selective of phosphoproteins directly immobilized on a plate.

Here, we have the same concept for the detection of using one of the most common techniques, Western blotting. pIMAGO molecule is synthesized to multiple titanium metal for selective binding to phosphates and biotin groups for sensitive After incubation with a where phosphoproteins are bound, the can be detected by chemiluminescence with peroxidase (HRP) conjugated to or by fluorescence. Under acidic the reagent is capable of specifically only to phosphorylated sites any bias toward amino sequences. Another feature of the reagent is the high ratio of groups per reagent, resulting in enhancement and amplification of the signal; even a protein with a low level can be detected. Without the of radioisotopes or expensive phospho-specific the technique is capable of detecting of interest phosphorylated under conditions, in the format of a standard blotting procedure. Alternatively, the can be functionalized with fluorescent instead of biotin to enable fluorescence-based detection.

Overall, the new displays superior selectivity phosphorylated proteins, enhanced and lower cost compared available commercial reagents. we demonstrate the utility of pIMAGO to phosphorylated proteins in simple and protein mixtures, as well as in vitro kinase and phosphatase We have further shown the technique is sensitive and specific to detect endogenous phosphorylation by analyzing Polo-like kinase 1 and its mutant protein complexes directly from cells. The were validated by quantitative spectrometry analysis, confirming a of known Plk1 substrates and proteins and identifying several new targets.

EXPERIMENTAL PROCEDURES

of Standard Proteins and pIMAGO-based

Membrane for dot-blot assays was in methanol, and different amounts of proteins were each in 0.5 μl of total volume. The membrane was to dry, followed by blocking 10 ml of SuperBlock T20 blocking buffer 1% BSA for 1 h. The membrane was then rinsed water and incubated for 1 h with 5 μl of the reagent (0.4 m m in 0.01% pH 5) in 10 ml of 500 m m glycolic acid, 1% TFA solution, pH The dot-blot was washed four with the 500 m m glycolic acid, 1% TFA and three times with 1× 5 min each wash. For detection, the was incubated with 1:5000 of avidin-peroxidase in 1× TBST (Tris-buffered containing 0.1% Nonidet pH 7.5) containing 1% BSA for 45 min while The membrane was lastly washed times with 1× TBST (5 min and detected with ECL detection


Cell Culture, Anti-Tyr(P) IP, and Dephosphorylation

HeLa cells grown to confluency in Dulbecco’s Eagle’s medium supplemented fetal bovine serum, 1% pyruvate, and 0.5% streptomycin/penicillin. The (6 × 10 7 ) were trypsinized, collected, and in 1 ml of lysis solution (50 m m Tris-HCl, pH 150 m m NaCl, 5 m m EDTA, 1% Nonidet 1 m m sodium orthovanadate, 1× protease mixture, 1× phosphatase inhibitor and 10 m m sodium fluoride) for 20 min on ice. The debris was cleared at 16,100 × g for 10 and supernatant containing soluble was collected. The concentration of the cell was determined using the bicinchoninic assay.

Two sets of 1 mg cell lysate incubated with 40 μl of anti-phosphotyrosine (clone PT66) conjugated to beads for 2 h at 4 °C. The beads were washed with the lysis and the bound proteins were with 100 m m triethanolamine. The elutions then dried and resuspended in 25 m m of buffer, pH 7.5. For dephosphorylation one-half of each elution was with 10 units of calf alkaline phosphatase (CIAP) in 1× buffer (50 m m Tris-Cl, 100 m m NaCl, 10 m m 2 . 1 m m dithiothreitol) for 30 min at 37 °C. To stop the enzyme the samples were boiled for 5 min in 1× SDS buffer.

In Vitro Phosphatase and Assays

For in vitro phosphatase 200 ng of ovalbumin and β-casein were with 2 units of CIAP in 1× buffer (50 m m Tris-Cl, 100 m m NaCl, 10 m m 2 . 1 m m dithiothreitol) for 30 min at 37 °C. Equal percentages of the solution were taken out at times and boiled for 5 min in 1× SDS sample The samples were run on an SDS gel, onto a PVDF membrane, and with pIMAGO as described After exposure, the membrane was using the stripping buffer m m Tris-Cl, 2% SDS, 0.7% pH 6.7) for 30 min at 65 °C and stained with Blue stain (0.1% Blue in the solution containing 10% acid, 45% methanol, 45% water) to the overall protein signal.

For in kinase assays, 200 ng each of or Cdc6 were incubated 500 μ m of ATP and 2 m m of MnCl 2 (for Syk) or 2 (for Cdk) in the presence of respective kinase (200 An equal amount of each solution was taken out at designated and boiled for 5 min in 1× SDS sample buffer. The were run on an SDS gel, transferred a PVDF membrane, and blotted pIMAGO as described before. detection of the Syk-band3 reaction pIMAGO, the membrane was stripped by with the stripping buffer m m Tris-Cl, 2% SDS, 0.7% pH 6.7) for 30 min at 65 °C. The membrane was then and incubated overnight at 4 °C with antibody (4G10 clone). washing and incubation with anti-mouse antibody for 45 min, the was exposed again to detect signal. Finally, both were stripped using the buffer and stained with blue stain to detect protein signal.

Detection of Phosphorylation of Plk1-interacting Proteins

HEK cells were transfected FLAG-Plk1 wild-type or K82M (kinase … mutant). transfection for 24 h, the cells were at mitosis by nocodazole (100 treatment for 12 h. For cells transfected FLAG-Plk1-K82M, BI 2536 (Plk1 100 n m ) was added for the last 8 h before Both sets of cells (5 × 10 7 were lysed in 500 μl of lysis (50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% P-40, 1 m m sodium orthovanadate, 1× inhibitor mixture (Sigma), 1× inhibitor mixture, 10 m m sodium for 20 min on ice. The cell debris was at 16,100 × g for 10 min, and supernatant soluble proteins was collected, in 4 mg of protein amount in each At this stage, a small of both lysates was stored at −20 °C for the blotting. The remaining lysates precleared with 20 μl of protein beads for 20 min at 4 °C, and the supernatant was collected. For IP, the samples were incubated for 2 h 40 μl of anti-FLAG beads at 4 °C. Finally, the was discarded, the beads were twice with the lysis for 10 min each, and 25% of the beads were to elute the bound proteins by in 2× SDS solution containing 40 m m DTT for 5 min at 95 °C. The resulting (including the set-aside lysates) run on an SDS gel (40 μg of lysates or one-half of IP elutions), onto a PVDF membrane, and with pIMAGO as described After the detection with the membrane was stripped by incubation the stripping buffer (62.5 m m 2% SDS, 0.7% β-mercaptoethanol, pH for 30 min at 65 °C. The membrane was then blocked and overnight at 4 °C with anti-phosphothreonine After washing and incubation secondary anti-rabbit antibody for 45 the membrane was exposed again to anti-Thr(P) signal. Finally, the was stripped again using the buffer and blotted for 1 h with antibody at room temperature. The Plk1 signal was detected incubation with secondary antibody for 45 min.

For the detection of pIMAGO signal fluorescence, IRDye 680 fluorophore-tagged was used instead of avidin-HRP. In case, the incubation was carried out same conditions as before, the detection was done out using Odyssey infrared imager, set at the channel of 700 nm and the intensity setting of

Mass Spectrometry-based Phosphoproteomic

The remaining 75% of the beads left anti-FLAG immunoprecipitation (from ∼3 mg protein amount) were for the phosphoproteomics experiment. The bound were eluted off beads, and reduced by incubating the beads in 50 m m bicarbonate containing 0.2% and 10 m m dithiothreitol for 5 min at 95 °C. After collecting the (eluted proteins), the samples cooled to room temperature and with 30 m m iodoacetamide for 1 h in the dark to the cysteines. The pH was adjusted to 8.0, and the were digested with 1 μg for 14 h at 37 °C. Following digestion, RapiGest was by decreasing pH to below 3.0 with 1 n acid, incubating the samples at 37 °C for 40 centrifuging it down for 10 min at 16,100 × g . and the supernatant. The resulting digested were then enriched by method to isolate phosphopeptides, as before (25 ).

The eluted phosphopeptides dried and redissolved in 8 μl of 0.5% acid. At this stage, 100 of 220 peptide library was added to sample as internal standard to label-free quantitation and injected an Eksigent two-dimensional Ultra HPLC system. The reverse C18 was performed using an in-house C18 column packed with 5 μm C18 beads resin (Michrom; inner diameter and 30 cm of bed length). The phase buffer consisted of HCOOH in ultra-pure water the eluting buffer of 100% CH 3 CN run a shallow linear gradient 60 min with a flow rate of 0.3 The electrospray ionization emitter tip was on the prepacked column with a puller (model P-2000; Instrument Co.). The Eksigent HPLC system was coupled with a high resolution linear ion trap Orbitrap spectrometer (LTQ-Orbitrap Velos; Fisher). The mass spectrometer was in the data-dependent mode in which a MS (from m / z 300–2000 with the of 30,000) was followed by 20 MS/MS of the most abundant ions. with charge state of +1 excluded. The mass exclusion was 90 s. The LTQ-Orbitrap raw files were directly against the Homo database with no redundant (91,464 entries; human Protein Index v.3.87) a combination of SEQUEST algorithm and on Proteome Discoverer (version Thermo Fisher). Peptide mass tolerance was set at 10 ppm, and tolerance was set at 0.8 Da. Search criteria a static modification of cysteine of +57.0214 Da and a variable modifications of Da to include potential oxidation of and a modification of +79.996 Da on serine, or tyrosine for the identification of phosphorylation. were performed with tryptic digestion and allowed a of two missed cleavages on the peptides from the sequence database. discovery rates were set 1% for each analysis. Proteome generates a reverse “decoy” from the same protein and any peptides passing the initial parameters that were from this decoy are defined as false positive The minimum cross-correlation factor was then readjusted for each charge state separately to meet the predetermined target discovery rate of 1% based on the of random false-positive matches the reversed “decoy” database. each data set had its own passing The most likely phosphorylation localization from collision-induced mass spectra was determined by algorithm within the Proteome 1.3 software.

Phosphopeptides identified by Mascot and with a statistical significant were quantified by in-house (manuscript in preparation). MASIC (27 ) in the software uses the m / z of the selected to construct extracted ion chromatograms default settings. Windows for ion chromatogram construction were 5 The relative intensity value of peptide was calculated by the peak of each individual extracted ion normalized to the total chromatogram of 220 equally spiked-in peptides Two identical peptides that a significant intensity ratio, is more than 3 or less 0.33, between two samples are to have different phosphorylation At least two biological replicates performed for the analysis.

The data with this manuscript, the MS/MS spectra and the raw MS files, may be from ProteomeCommons.org Tranche, using the following hash KSeBmSNefSWKnYB66DFXtpMT9So8CtRf5xZ0U5PQl6C4AtYsS1eKbdsqXaskdYMb2IcECd/x05fo4A6a44OJQMkZsmsAAAAAAAAEIQ==.

RESULTS

The design of is based on a soluble nanopolymer, . dendrimer, with hyperbranched groups for derivatization. In addition to solubility under aqueous the advantages of using soluble include high structural and homogeneity, compact spherical and controlled surface functionalities (28 ). The and hyperbranched nature of the functionalized exhibited high reactivity and specificity toward phosphorylated (25 ).

Polo vivo

A schematic representation of the pIMAGO A . a soluble nanopolymer (dendrimer) is with multiple titanium for selective binding to the phosphoproteins and biotin groups for sequential by avidin-linked HRP. B . experimental for pIMAGO-based phosphoprotein detection in a blotting format. After of the protein mixture and transfer a PVDF membrane, the blot is incubated with the pIMAGO which selectively binds to HRP-linked avidin is then to the membrane, exclusively attaching to the groups of pIMAGO. Finally, a HRP substrate is added, and the membrane is for detection.

Fig. 2.

pIMAGO-based of phosphoproteins. A . a dot-blotting experiment, two phosphoproteins (α-casein and ovalbumin) and two (α-lactalbumin and β-lactoglobulin) were on a PVDF membrane and detected pIMAGO procedure. B . a dot-blotting where phosphorylated α-casein and nonphosphoproteins (α-lactalbumin, β-lactoglobulin, and catalase) were spotted at amounts on PVDF membranes and using pIMAGO procedure. C . detection of different concentrations of the mixture separated by SDS-PAGE and onto a PVDF membrane. The included phosphorylated α-casein and nonphosphoproteins (α-lactalbumin, β-lactoglobulin, and catalase).

To further test the and sensitivity of the pIMAGO technology, we five standard proteins in amounts, ranging from 10 to 100 ng, for a dot-blot analysis. The proteins phosphorylated α-casein and four proteins (BSA, catalase, and β-lactoglobulin). The results, shown in 2 B . demonstrate good selectivity and of the blotting reagent, allowing of only phosphorylated α-casein, below 10 ng levels.

We have explored the ability of pIMAGO to detect phosphoproteins in a Western format by mixing together the five proteins at different separating the mixture on a gel, and onto a PVDF membrane. As in Fig. 2 C . pIMAGO-based blotting was able to detect only the with good sensitivity, the background was slightly higher the dot blotting because of the presence of SDS on the after gel transfer, which led to lower sensitivity. Taken these data suggest pIMAGO could be a valuable for the detection of phosphorylation after separation and transfer.

Additional of the blotting selectivity was carried out a more complex sample, a mixture isolated by anti-phosphotyrosine immunoprecipitation (anti-Tyr(P) IP). 1 mg of HeLa lysate as the starting the resulting protein complex was equally in halves. One sample was with a general CIAP. Two were run alongside on the SDS-PAGE transferred onto a membrane, and independently using either or anti-phosphotyrosine antibody. The results a significant decrease in signal the phosphatase-treated samples in both indicating that pIMAGO is specific for phosphoproteins in a complex (Fig. 3 ). Interestingly, proteins a different pattern when by pIMAGO compared with the anti-Tyr(P) antibody approach: a of new bands were detected the pIMAGO method. The new bands are due to the ability of pIMAGO to detect not phosphotyrosine proteins, which is the for the anti-Tyr(P) antibody, but also and phosphothreonine residues. It is also that some of the new protein are phosphotyrosine proteins that be detected by this particular of anti-Tyr(P) antibody. The experiment that pIMAGO can be used to any phosphoprotein without the bias antibodies may have.

Fig. 3.

pIMAGO-based detection of a mixture of phosphoproteins. Cell was used to immunoprecipitate ( IP ) phosphotyrosine-containing ( pY ). One-half of each IP elution was by general CIAP. Finally, the and CIAP-treated samples were in two, separated by SDS-PAGE, onto a membrane, and detected using pIMAGO or anti-phosphotyrosine

Because pIMAGO has demonstrated its to detect any phosphorylation without it would be particularly useful for the of changes in phosphorylation during in kinase and phosphatase assays. procedures, although routine, can invaluable knowledge about the or phosphatase-substrate relations and can be used to or validate new interesting substrates. such an approach requires preliminary knowledge of the phosphorylation in the case of antibody-based detection; the use of substrates, which could misleading information; or handling of the hazardous radioactive materials, in the of 32 P-based reactions. To address the and high cost of the antibodies and the concerns of the radioactive assays, we to examine whether pIMAGO is of detecting changes in phosphorylation in vitro kinase or phosphatase

Fig. 4.

In vitro phosphatase and assays using pIMAGO. A . two (β-casein and ovalbumin) were with CIAP for the indicated Each assay time was run on the SDS gel, transferred onto a and blotted by pIMAGO. B . in vitro assay of Syk tyrosine kinase and its band3 was carried out for the indicated Phosphorylation change on the substrate was by pIMAGO; the membrane was then and reprobed with anti-phosphotyrosine C . in vitro kinase assay of Cdk kinase and its substrate Cdc6 was out for the indicated times. Phosphorylation on the substrate was detected by pIMAGO. In all Coomassie stain was performed to equal loading.

We further out in vitro kinase assays a purified tyrosine kinase, Syk, and a serine/threonine kinase, Cdk1, along with known substrates, band3 30 ) and Cdc6 (31 ), respectively. Each reaction was carried out for the designated points, and each assay mix was run on an SDS gel and For the Syk-band3 kinase assay, the was also blotted with a anti-Tyr(P) antibody after On the other hand, because no general anti-serine/threonine antibody is Cdk1-Cdc6 kinase reaction was only with pIMAGO. As is in Fig. 4 ( B and C ), both kinase resulted in increased levels of as was detected by pIMAGO and confirmed by antibody blotting. Coomassie was also carried out for the assays to equal loading.

Our experiments that pIMAGO has the potential to the often unnecessary or ineffective and phosphospecific antibodies and the hazardous labeling approach for routine activity assays. This has the potential for high sensitivity there are multiple biotin groups per molecule, thus the detection signal. The technique is appealing to research groups work with new or uncharacterized and their substrates, whose profiles are unknown. Instead of experiments to map potential phosphorylation of their substrates to make new antibodies, the pIMAGO reagent can be used for a majority of initial assays for any substrate.

In addition to the use of in the Western blot format for of in vitro phosphorylation, the technology offers great potential for in vivo or endogenous phosphorylation. To this idea, we have serine/threonine kinase Plk1 and its proteins as our model system. is a member of an important Polo-like family and is a well known key in many cell cycle As a serine/threonine kinase, Plk1 in mitosis is critical for proper of mitotic entry, cytokinesis, spindle formation, and sister separation (32 ⇓ –34 ). In addition, it has also suggested that Plk1 an important role in the network of DNA checkpoint (35. 36 ). Taking into account, it is not surprising numerous recent studies demonstrated a strong correlation mammalian Plk1 expression and (33. 37 ). Its overexpression has been documented in many carcinomas, non-small cell lung ovarian …, gastric melanoma, and others (33. 38 ). selective inhibition of Plk1 may provide an important complementary in … therapy. Plk1 has involved in multiple signaling and its tumorigenic features are tightly to its modulated phosphorylation events and partners (38 ). To better understand the of Plk1 in normal cells and progression, it is of critical importance to the protein signaling in the Plk1-dependent

To assess the phosphorylation levels of proteins, HEK 293T cells transfected with FLAG-tagged and arrested in mitosis where is most active. As a comparison, the were also transfected to a kinase … (KD) of Plk1, and BI 2536, a specific inhibitor (39. 40 ), was incubated these cells. Both of samples were immunoprecipitated anti-FLAG antibody, run on an SDS-PAGE, and via pIMAGO. Simultaneously, to validate the imaging results and identify the proteins obtained using detection system, we used 75% of the samples to run a quantitative phosphoproteomics using mass spectrometry workflow is demonstrated in Fig. 5 ). As both sets of lysates and IP showed high levels of (Fig. 6 A ). The phosphorylation signal of the Plk1 lysate was slightly compared with the Plk1-KD likely because of increased of the Plk1 kinase. Interestingly, IP of mutant clearly exhibited a level of phosphorylation in the interacting compared with wild-type As a comparison, the membrane was also using an anti-Thr(P) antibody, showed a similar phosphorylation although only of threonine (Fig. 6 B ). An anti-FLAG Western was further carried out as a loading (Fig. 6 C ). These data that the phosphorylation levels of proteins are higher in the kinase mutant of the kinase. The data the perception that kinase-substrate are typically weak and transient. substrates are phosphorylated, the interactions are weaker, leading to the dissociation of from the kinase. On the other KD mutant has stronger binding to its substrates because the post-activity does not occur, which can be captured by IP. These results that pIMAGO technique can an overall profile of phosphorylation in kinase complexes.

Experimental of pIMAGO detection and phosphoproteomic of Plk1-interacting proteins. FLAG-Plk1 WT or (kinase … mutant) transfected into HEK 293T and arrested in mitosis, and the cells lysed. The interacting protein for Plk1 were separately down by anti-FLAG antibody with beads. At this 25% of each sample were run on SDS-PAGE, transferred onto a and detected by pIMAGO. The remaining 75% trypsin-digested, and phosphopeptides were by PolyMAC method. Prior to spectrometry analysis, a peptide consisting of 220 unique peptides was into each sample for quantitation.

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