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1 Supplementary Materials for Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells Scott F. Rusin, Kate A. Schlosser, Mark E. Adamo, Arminja N. Kettenbach* This PDF file includes: *Corresponding author. Published 13 October 2015, Sci. Signal. 8, rs12 (2015) DOI: /scisignal.aab3138 Fig. S1. Characterization of PP6c shrnas. Fig. S2. Quantitative comparison of protein abundance changes upon PP6c depletion with individual PP6c shrnas. Fig. S3. Scheme of cell cycle synchronization. Fig. S4. Characterization of PP6c-regulated phosphopeptides. Fig. S5. LC-MS/MS trace and MS/MS spectra of known PP6c substrate DNA-PK pser Fig. S6. Interaction network of proteins with increased and decreased phosphorylation upon PP6c depletion. Fig. S7. LC-MS/MS trace and MS/MS spectra of NCAP-G pser 973/5. Fig. S8. Characterization of PP6c and NCAP-G purifications. Fig. S9. NCAP-G analysis. Legend for tables S1 to S8 Other Supplementary Material for this manuscript includes the following: (available at Table S1 (Microsoft Excel format). Table containing TMT protein quantification results. Table S2 (Microsoft Excel format). Table containing significantly increased phosphopeptides due to phosphorylation or protein abundance, significantly decreased phosphopeptides due to phosphorylation or protein abundance, and all phosphopeptide and protein data.
2 Table S3 (Microsoft Excel format). Table containing quantification results of AURKA substrates. Table S4 (Microsoft Excel format). Table containing quantification results of RXS motif containing phosphopeptides that increase in phosphorylation occupancy upon PP6c depletion. Table S5 (Microsoft Excel format). Table containing proteins identified in 3 Flag- NCAP-H and 3 Flag-PP6c purification. Table S6 (Microsoft Excel format). Table containing quantification results of the PP6c in vitro phosphatase assays of NCAP-G. Table S7 (Microsoft Excel format). Table containing quantification results of the CK2 in vitro kinase assay of NCAP-G. Table S8 (Microsoft Excel format). Table containing quantification results of proteinase K digest of NCAP-G.
3 Figure S1. Characterization of PP6c shrnas. (A) Quantification of GFP expression in HeLa cells uninfected or infected with individual PP6c shrnas. (B) Western blot analysis of PP6c and GFP abundance in HeLa cells uninfected or infected with individual PP6c shrnas. Lamin A/C loading control. (C) Quantification of PP6c abundance normalized to Lamin A/C (n = 3 independent experiments). ** p-value less than (D) Western blot analysis of PP6c, GFP, and AURKA pthr 288 abundance in mitotically-arrested HeLa cells infected with control, individual PP6c-sh1 and PP6c-sh4, and combined PP6c-sh1+4. (E) Quantification of PP6c, AURKA pthr 288 and GFP abundance normalized to Lamin A/C. ** p-value less than 0.01, *** p-value less than 0.001, (n = 3 independent experiments). Note: AURKA pthr 288 is only significantly increased upon depletion of PP6c below 10% abundance compared to control infected HeLa cells.
4 Figure S2. Quantitative comparison of protein abundance changes upon PP6c depletion with individual PP6c shrnas. (A) Scheme depicting experimental strategy of HeLa cell infection and cell cycle synchronization. HeLa cells infected with control, PP6csh1, or PP6c-sh4 baculoviruses were synchronized in their cell cycle progression at the G1/S-phase boundary using thymidine followed by wash-out and arrest in mitosis with the microtubule stabilizing drug Taxol. Mitotically-arrested HeLa cells were collected, separately lysed, reduced, alkylated, and trypsin digested. Peptides were isotopically-labeled with isobaric tandem-mass-tag (TMT) reagents, separated by reverse phase chromatography, and analyzed by MS 3 -quantitative LC-MS/MS on an Orbitrap Fusion mass spectrometer (n = 3 independent experiments). The experiment was performed in biological triplicates for each condition. (B) Scatter plot of average log 2 ratios of PP6c-sh1 versus control shrna and PP6c-sh4 versus control shrna protein fold-changes. Note: out of 6700 identified and quantified proteins, only 35 are significantly different between the two shrnas (indicated in red) with a maximum absolute log 2 fold-change of Protein ratios corresponding to PP6c and the three PPP6R regulatory subunits are indicated on the plot.
5 Figure S3. Scheme of cell cycle synchronization. Scheme depicting experimental strategy of HeLa cell infection and cell cycle synchronization. HeLa cells were infected with control or PP6c shrna baculoviruses and synchronized in their cell cycle progression at the G1/S-phase boundary using thymidine, followed by thymidine wash-out and arrest in mitosis with the microtubule stabilizing drug Taxol. Mitotically-arrested HeLa cells were collected, separately lysed, reduced, alkylated, and trypsin digested. Figure S4. Characterization of PP6c-regulated phosphopeptides. (A) Diagram depicting data analysis workflow for PP6c-dependent phosphorylation changes. (B) Bar graph depicting number of significantly regulated phosphopeptides per protein upon PP6c depletion.
6 Supplementary Figure 5. LC-MS/MS trace and MS/MS spectra of known PP6c substrate, DNA-PK pser 3205 A, Relative intensities of LC-MS/MS traces of heavy- and light-labeled phosphopeptides covering Ser 3205 phosphorylation site on DNA-PK, a previously described PP6c substrate site, extracted to +/- 2 ppm. Phosphopeptide sequence, charge state, and ion mass-tocharge values are indicated. B, Annotated MS/MS spectrum of the phosphopeptide corresponding to DNA-PK Ser 3205.
7 Figure S6. Protein interaction network of proteins with increased and decreased phosphorylation upon PP6c depletion. (A) Protein-protein interaction network of proteins containing phosphopeptides significantly increased in phosphorylation upon PP6c depletion generated with STRING. Node colors correspond to highly connected clusters. (B) Protein-protein interaction network of proteins containing phosphopeptides significantly increased in phosphorylation upon PP6c depletion generated with STRING. Node colors correspond to highly connected clusters.
8 Supplementary Figure 7. LC-MS/MS trace and MS/MS spectra of NCAP-G pser 973/5 A, Relative intensities of LC-MS/MS traces of heavy- and light-labeled phosphopeptide covering pser 973/5 phosphorylation site on NCAP-G, extracted to +/- 2ppm. Phosphopeptide sequence, charge state, and ion mass-to-charge values are indicated. B, Annotated MS/MS spectrum of the phosphopeptide corresponding to NCAP-G pser 973/5 as identified in the large-scale shpp6 screen.
9 Figure S8. Characterization of PP6c and NCAP-G purifications. (A) Coomassie gel of 3xFlag-NCAP-H and 3xFlag PP6c purifications. Condensin I subunits, PP6c, SAPS, and ANR subunits, as well as the endogenous inhibitor α4 are indicated. Bovine serum albumin (BSA) standard amounts are provided for quantitative reference. (B) ibaq analysis of PP6c purifications. 3xFlag-PP6c was purified, acid precipitated, and protein abundance was quantified (n = 3 independent experiments). Note: the majority of this PP6c is bound to either of the two known endogenous inhibitors α4 and TIPRL. Figure S9. NCAP-G analysis. Frequency plot depicting log 2 ratio distribution of Proteinase K digested NCAP-G peptides.
10 Supplementary Tables provided as Excel files. Table S1. Table containing TMT protein quantification results. Table S2. Table containing significantly increased phosphopeptides due to phosphorylation or protein abundance, significantly decreased phosphopeptides due to phosphorylation or protein abundance, and all phosphopeptide data and protein data. Table S3. Table containing quantification results of AURKA substrates. Table S4. Table containing quantification results of RXS motif containing phosphopeptides that increase in phosphorylation occupancy upon PP6c depletion. Table S5. Table containing proteins identified in 3 Flag-NCAP-H and 3 Flag-PP6c purification. Table S6. Table containing quantification results of the PP6c in vitro phosphatase assays of NCAP-G. Table S7. Table containing quantification results of the CK2 in vitro kinase assay of NCAP-G. Table S8. Table containing quantification results of proteinase K digest of NCAP-G.
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