EMBO/SourceData huggingface dataset:
Figure legend: "(D) The MS orientation was determined in metaphase cells in organoids of Caco-2 cells transfected with control (n=75), ALIX (n=88) or NuMA (n=79) siRNA (from three independent experiments). The graph shows the average percentage of Caco-2 cells with the indicated relative spindle angle (±S.E). Greater spindle angle variation was observed in ALIX- and NuMA-depleted cells compared to control cells (***p&lt;0.001, ANOVA). Representative confocal images are shown where the cells are stained with rhodamine-phalloidin (red), α-tubulin (green) and Hoechst (blue). The apical membrane facing the lumen of the organoid is marked (*). Scale bar, 5 μm"
NCBI gene IDs:
"NuMA: 4926
ALIX: 10015"
Figure legend: "(E) Lysates of control, ALIX or NuMA-depleted Caco-2 cells were immunoblotted for NuMA, ALIX, α-tubulin and GAPDH (loading control)"
NCBI gene IDs:
"NuMA: 4926
ALIX: 10015"
Figure legend: " (A) The MARCM strategy was used to generate alix mutant NBs clones (FRT82B, alix1) or genetic equivalent control clones (FRT82B con) in the larval brain. Dissected brains were immunostained with anti-α-tubulin (red) and Hoechst (blue). Recombinant NBs are positively marked with GFP (green) and outlined. The presence or lack of astral MTs is indicated with arrows or asterisks, respectively. Scale bars, 5 μm. The average intensity of astral, total and astral/total MT (±S.E) in GFP-positive relative to GFP-negative control cells (set to 100%, indicated as dotted line in the graph) was calculated from three separate experiments (including 13 GFP-negative FRT82B con NBs, 11 GFP-positive FRT82B con NBs, 10 GFP-negative FRT82B, alix1 NBs and 8 GFP-positive FRT82B, alix1 NBs) (*p&lt;0.05 and **p&lt;0.01, Student's t-test) "
NCBI gene IDs:
"alix: 43330"
Figure legend: "(B) HeLa cells depleted of ALIX or not were immunostained with anti-α-tubulin (red), anti-Pericentrin (green) and Hoechst (blue). Representative confocal images are presented. Scale bars, 5 μm. The graph shows MT intensities as percentage of control cells (±S.E) obtained from 99 control and 90 ALIX siRNA transfected cells (data obtained from six separate experiments). Significant differences are noted as *p&lt;0.05 and ***p&lt;0.001 (Student's t-test)"
NCBI gene IDs:
"ALIX: 10015"
Figure legend: "(C, D) Regrowth of MTs after cold-induced depolymerization was performed using brains of w1118 and alix1 larvae, stained with anti-α-tubulin (red), anti-Bazooka (green) and Hoechst (blue) (C Typical confocal images showing MT-regrowth at given time points are shown (scale bars, 5 μm). The graphs show the time-dependent MT intensities relative to the respective 0''-samples (±S.E) calculated from three independent experiments. In (C) 46, 64, 65 and 48 w1118 NBs and 73, 37, 44 and 49 alix1 NBs from 0, 30, 60 and 300'' were analyzed, respectively (*p&lt;0.05)"
NCBI gene IDs:
"alix: 43330"
Figure legend: "(C, D) Regrowth of MTs after cold-induced depolymerization was performed usin HeLa cells depleted of ALIX or not stained with anti-α-tubulin (white) and anti-Pericentrin (green) (D) Typical confocal images showing MT-regrowth at given time points are shown (scale bars, 5 μm). The graphs show the time-dependent MT intensities relative to the respective 0''-samples (±S.E) calculated from three independent experiments In (D) 47, 40 and 42 control cells and 48, 47 and 47 ALIX-depleted cells were calculated 0, 30'' and 420'' after regrowth, respectively (*p&lt;0.05)"
NCBI gene IDs:
"ALIX: 10015"
Figure legend: "(E) Brains dissected from control (FRT82B con) or alix1 (FRT82B, alix1) larvae were immunostained with anti-γ-tubulin (red) and Cnn (white, left panel) or anti-Dgrip91 (red) and α-tubulin (white, right panel), as well as Hoechst (blue). Recombinant NBs are positively marked with GFP (green). The confocal images show typical accumulation of centrosomal γ-tubulin or Dgrip91, and the insets zoom in at the indicated centrosomes. Scale bars, 5 μm. The intensities of γ-tubulin, Dgrip91 and Cnn in control NBs (GFP-negative, set to 100 %) and GFP-positive, recombinant NBs are presented in the table (±S.E), also indicating the number of NBs quantified (n) from three separate experiments (*p&lt;0.05)"
NCBI gene IDs:
"alix: 43330"
Figure legend: "(H) HeLa cells depleted of ALIX or not grown on coverslips were stained with anti-γ−tubulin (white), anti-Pericentrin (red) and Hoechst (blue) before being examined by confocal fluorescence microscopy. The confocal images and insets show the centrosomal accumulation of γ-tubulin and Pericentrin. Scale bars, 5 μm. The graph presents the average centrosomal intensities of γ-tubulin and Pericentrin relative to control cells (set to 100%) calculated from three independent experiments (10 cells per condition/experiment) (±S.E). Significant difference is noted as **p&lt;0.01 (Student's t-test)"
NCBI gene IDs:
"ALIX: 10015"
Figure legend: "(I) Lysates of control or ALIX-depleted HeLa cells were immunoblotted for ALIX, γ-tubulin, α-tubulin and GAPDH (loading control)"
NCBI gene IDs:
"ALIX: 10015"
Figure legend: " (A) The MS orientation was determined in metaphase NBs from brains dissected from wild type (w1118), alix1, γ−Tub23CPI or γ−Tub23CPI; alix1 larvae. Compared to w1118, significantly greater variation in the relative spindle angles was observed in alix1 NBs and γ−Tub23CPI; alix1 NBs (*p&lt;0.05, ANOVA). The γ−Tub23CPI;alix1 NBs displayed significantly greater variation of their spindle orientations than either alix1 or γ−Tub23CPI NBs (*p&lt;0.05, ANOVA). The graph shows the average percentage of NBs with the indicated relative spindle angles (±S.E) from in total 57 w1118 NBs, 55 alix1 NBs, 76 γ−Tub23CPI NBs and 62 γ−Tub23CPI; alix1 NBs (three independent experiments) "
NCBI gene IDs:
"alix: 43330
γ−Tub23C: 33501"
Figure legend: "(C) Dissected brains of w1118, alix1, γ−Tub23CPI and γ−Tub23CPI;alix1 larvae were subjected to MT regrowth assay and stained with anti-α-tubulin (red), anti-Cnn (white) and Hoechst (blue). Representative images of NBs after 0 and 60 seconds regrowth are presented. Scale bars, 5 μm"
NCBI gene IDs:
"alix: 43330
γ−Tub23C: 33501"
Figure legend: "(D) The MT-intensities after 0 and 60 seconds regrowth were determined, here presented as the average of three separate experiments (± S.E) where in total 27 w1118-0'', 28 w1118-60'', 26 alix1-0'', 19 alix1-60'', 28 γ−Tub23CPI-0'', 26 γ−Tub23CPI-60'', 28 γ−Tub23CPI;alix1-0'' and 23 γ−Tub23CPI;alix1-60'' NBs were analyzed. Compared to w1118-NBs, significantly less MT polymerized in alix1, γ−Tub23CPI or γ−Tub23CPI;alix1 NBs (*p&lt;0.05, ANOVA)"
NCBI gene IDs:
"alix: 43330
γ−Tub23C: 33501"
Figure legend: "(E) The relative spindle angle between the polarity axis perpendicular to the apical phalloidin-stained F-actin surface and the MS was determined in metaphase Caco-2 cysts cells transfected with control, ALIX and/or γ-tubulin siRNA (29, 29, 18 and 32 metaphase cells, respectively, were analyzed from three separate experiments). Compared to control cells, depletion of either ALIX or both ALIX and γ-tubulin, induced a significantly increased randomization of the relative spindle angles (**p&lt;0.01 and ***p&lt;0.001, ANOVA). Caco-2 cells depleted of ALIX+γ-tubulin showed a greater variation in the spindle angles compared to depletion of ALIX or γ-tubulin (*p&lt;0.05, ANOVA). ALIX-depleted cells showed greater spindle angle-variation than γ-tubulin-depleted cells (*p&lt;0.05, ANOVA)"
NCBI gene IDs:
"ALIX: 10015
γ-tubulin: 7283"
Figure legend: " (A) HeLa cells transfected with control or ALIX siRNA were fixed, permeabilized and stained with anti-MAP1S (red), anti-γ-tubulin (white) and Hoechst (blue). Interphase cells are shown and the insets show close ups of the centrosomes. Scale bars, 5 μm. (B) The fluorescence intensity of centrosomal MAP1S was determined and the graph shows the average of three independent experiments (± S.E), in which in total 52 control and 36 ALIX siRNA cells were analyzed. Significant difference is indicated as *p&lt;0.05 (Student's t-test) "
NCBI gene IDs:
"ALIX: 10015"
Figure legend: "(C) HeLa cells were transfected with control, ALIX, MAP1S or ALIX+MAP1S siRNA and the expression of ALIX, MAP1S and GAPDH (loading control) was determined by Western blotting analysis. Representative results are presented"
NCBI gene IDs:
"MAP1S: 55201
ALIX: 10015"
Figure legend: "(G) HeLa cells transfected with control or ALIX siRNA grown on coverslips were transiently transfected with pEYFP-C1 (green), pEYFP-MAP1S (green), pEGFP-C1 (green) or pEGFP-CLASP2α and immunostained with anti-α-tubulin (white), anti-Pericentrin (red) and Hoechst (blue). Scale bars, 5 μm"
NCBI gene IDs:
"CLASP2α: 23122
MAP1S: 55201
ALIX: 10015"
Figure legend: "(H) The lengths and numbers of astral MTs of HeLa cells treated as in (G) were measured (LSM software, version 3.2). The graphs show the average numbers (±S.E) based on calculating 30 control siRNA+YFP cells, 23 control siRNA+YFP-MAP1S cells, 30 ALIX siRNA+YFP cells, 30 ALIX siRNA+YFP-MAP1S cells, 28 control siRNA+GFP cells, 30 control siRNA+GFP-CLASP2α cells, 30 ALIX siRNA+GFP cells and 29 ALIX siRNA+GFP-CLASP2α cells from three separate experiments (*p&lt;0.05, Student's t-test), respectively"
NCBI gene IDs:
"CLASP2α: 23122
MAP1S: 55201
ALIX: 10015"
Figure legend: " Mean IC50 values (µM) for APR-246 +/- 20 μM MK-571 of 21 cell lines. *p &lt; 0.0001, Wilcoxon test. Each dot represents one cell line. Mean IC50 values and n shown in Table S1. ZIP synergy scores of most synergistic area sub-grouped according to TP53 gene status for 21 cell lines. Each dot represents one cell line. Scores and n are shown in Table S1. "
NCBI gene IDs:
"TP53: 7157"
Figure legend: "Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53."
NCBI gene IDs:
"MRP1: 4363
p53: 7157"
Figure legend: "Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (n ≥ 5 and n ≥ 2 respectively) after 48h APR-246 treatment. Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs. Data and n of individual siRNAs are shown in Fig S1J, data is also part of Fig 7B."
NCBI gene IDs:
"MRP1: 4363"
Figure legend: "Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector (EV) and MRP1 expression vector."
NCBI gene IDs:
"MRP1: 4363"
Figure legend: "Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with empty vector (EV) and MRP1 vector after 72h APR-246 treatment (n = 4). Data is also part of Fig S7E."
NCBI gene IDs:
"MRP1: 4363"
Figure legend: "Growth of Eso26 xenografts (R248W mutant TP53) in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. p values determined by one-way ANOVA with Tukey's correction at day 18, n is indicated in the figure.   Kaplan-Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. Mice that were culled at ethical endpoints prior to reaching a tumor volume of &gt;1400mm3 are marked as censored observations. p values were determined by Log-rank (Mantell-Cox) test between different treatment groups. n is indicated in the figure.   "
NCBI gene IDs:
"TP53: 7157"
Figure legend: "14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells (n ≥ 3 and ≥ 2) transfected with MRP1 siRNAs after 24h of 14C-APR-246 treatment. Indicated values represent four individual MRP1 siRNAs and two individual negative control (Ctrl) siRNAs, where each dot is an individual siRNA. Mean of 14C-accumulation after treatment and n with individual siRNA sequences is shown in Table S4. *p = 0.04 and for R248W p = 0.06, Wilcoxon matched pairs signed rank test."
NCBI gene IDs:
"MRP1: 4363"
Figure legend: "APR-246 content in OVCAR-3 cells (TP53 R248Q) 24h after treatment with APR-246 +/- MK-571, (n = 3). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4."
NCBI gene IDs:
"TP53: 7157"
Figure legend: "Mean IC50 (µM) values of APR-246 in 17 different cell lines (n ≥ 3) as shown by WST-1. *p = 0.03, Mann Whitney test. Mean IC50 (µM) values for APR-246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5."
NCBI gene IDs:
"TP53: 7157"
Figure legend: "Box-and-whisker plot of Pearson correlations between PRIMA-1 area-under-the-curve (AUC) and the levels of 225 different metabolites from the DepMap portal. Central band indicates median, boxes indicate 25th and 75th percentile, and whiskers show 2.5th and 97.5th percentile outlier metabolites. High GSH and GSSG (in red) correlate with low PRIMA-1 sensitivity. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5."
NCBI gene IDs:
"TP53: 7157"
Figure legend: " Total intracellular glutathione (GSH+GSSG) as shown by glutathione reductase (GR) re-cycling assay in H1299 R175H (tet-off) (+/- doxycycline) and H1299 -/- cells 24h after seeding (n = 3). Indicated values are fold change compared to mutant TP53 cells. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. "
NCBI gene IDs:
"TP53: 7157"
Figure legend: " Total intracellular glutathione (GSH+GSSG) as shown by GR re-cycling assay in untreated HCT116 (R248W, WT and -/-) cells 48h after seeding (n = 3, 5 or 2 respectively). Indicated values are fold change to mutant TP53 cells. Part of this data is shown in Fig. EV4. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. "
NCBI gene IDs:
"TP53: 7157"
Figure legend: "Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells (n = 2) Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5."
NCBI gene IDs:
"TP53: 7157"
Figure legend: " Correlation between 14C accumulation (cpm/mg x ml-1) after treatment with 25 µM 14C-APR-246 and IC50 (µM) values for APR-246 in 11 cell lines (n ≥ 3) (r = -0.66, p = 0.03, Pearson correlation). Each dot represents one cell line, 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. "
NCBI gene IDs:
"TP53: 7157"
Figure legend: " ABCC1 mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) in lung cancer (luad &amp; lusc studies, *p&lt;0.0001) and colon adenocarcinoma (coad study, *p=0.005) grouped into having no alterations or putative driver missense mutations in TP53. Statistical analysis by Mann Whitney test, n is indicated in the figure. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. "
NCBI gene IDs:
"ABCC1: 4363
TP53: 7157"
Figure legend: " 14C accumulation (cpm/mg x ml-1) in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells after 24h treatment with 14C-APR-246 (n = 2). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. "
NCBI gene IDs:
"TP53: 7157"
Figure legend: " Growth suppression in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells treated for 72h with APR-246 as shown by WST-1 assay (n = 3). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. "
NCBI gene IDs:
"TP53: 7157"
Figure legend: "Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3)."
NCBI gene IDs:
"TP53: 7157"
Figure legend: " 14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W mutant TP53 cells +-transfected with MRP1 or xCT siRNA following 24h of 14C -APR-246 treatment +/- MK-571 . Indicated values are mean values from four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n = 1-2) and two control siRNAs (n ≥ 3). Mean 14C -accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C -APR-246 treatment is also shown in Fig 3D. "
NCBI gene IDs:
"MRP1: 4363
xCT: 23657
TP53: 7157"
Figure legend: " Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR-246 treatment as shown by WST-1 assay. Indicated values are mean values of four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n =1-2) and two control siRNAs (n ≥ 3). Data for control and MRP1 siRNA after APR-246 single treatment are also included in Fig 1I. Values and n for individual siRNA shown in Fig. S7C. "
NCBI gene IDs:
"MRP1: 4363
xCT: 23657"
Figure legend: " Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT cells as shown by GR re-cycling assay 48h post transfection of siRNA. Indicated values are means from four MRP1 siRNAs (n = 3), two xCT siRNA (n = 2-3) and two control siRNA (n = 2-3). "
NCBI gene IDs:
"MRP1: 4363
xCT: 23657"
Figure legend: " OVCAR-3 (R248Q TP53) confluence by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalzine (SSZ) (n = 3). Part of the data are shown in Fig 1G. Growth suppression in OVCAR-3 cells as determined by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ) depicted as heatmaps (n = 3). "
NCBI gene IDs:
"TP53: 7157"
Figure legend: "(A) qRT-PCR analysis of SUMO-modifying proteins and enzymes in cortex and striata of WT mice at 12 weeks. Relative expression for all the SUMO enzymes is normalized to mouse β-actin."
NCBI gene IDs:
"β-actin: "
Figure legend: "(B and C) qRT-PCR of SUMO mRNAs from 12-week-old WT and R6/2 mouse cortex (B) and striatum (C). SUMO enzyme mRNAs are differentially expressed in R6/2 versus control with statistically significant increases in SENP1 (p = 0.01), SENP3 (p = 0.02), and SUMO-1 (p = 0.003) in cortex and SENP1 (p = 0.02), SENP6 (p = 0.04), PIAS3 (p = 0.007), SUMO-1 (p = 0.02), and SUMO-2 (p = 0.02) in striatum. Samples were analyzed in quadruplicate and normalized to mouse β-actin. Data are shown as R6/2 expression relative to WT levels set at 1 for each enzyme with ± SD (n = 4). *p &lt; 0.05. n.s., not significant."
NCBI gene IDs:
"β-actin: 
PIAS3: 229615
SENP1: 223870
SENP3: 80886
SENP6: 215351
SUMO-1: 22218
SUMO-2: 170930"
Figure legend: "(A) HeLa cells transfected with His-tagged HTTex1p (46Q) or 46QP-K6,9,15R (3R) along with GFP-SUMO-1 or GFP-SUMO-2, lysed under denaturing conditions, and nickel purified (Ni-NTA). Unmodified HTT-46Q is indicated by the arrow and SUMO-modified HTT by the boxed region. The lysine mutant (3R) serves as a negative control. Ni-NTA represents nickel-purified His-tagged HTT, and WC TCA represents 10% of the whole-cell lysate expression of HTT and myc-actin (transfection control). HTT is modified by SUMO-1 (left) and SUMO-2 (right)."
NCBI gene IDs:
"HTT: 3064
SUMO-1: 7341
SUMO-2: 6613"
Figure legend: "(B) SUMO isopeptidases (SENP1, SENP2, SENP3, SENP5, and SENP6) modulate SUMO-modified HTT when overexpressed together with HTT (46QP-H4 or 3R) and SUMO-1 (GFP-SUMO-1). SENP1, SENP2, and SENP6 decrease HTT SUMOylation. Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System (LI-COR) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100."
NCBI gene IDs:
"HTT: 3064
SENP1: 29843
SENP2: 59343
SENP3: 26168
SENP5: 205564
SENP6: 26054"
Figure legend: "(C) Titration of SUMO-1. Denaturing nickel purification of HTTex1p (46QP-H4) following transfection with decreasing amounts of SUMO-1 reduces the amount of SUMO-modified HTT to undetectable levels. The Ni-NTA blot displayed in the gray scale shows purified HTTex1 and SUMO-modified HTTex1 using HTT antibody. Note that 0.5 μg of SUMO-1 (¼ the amount of SUMO-1 cDNA ) was used for identifying the SUMO-1 E3 ligase for HTTex1p. Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100. Note that all experiments were performed in triplicate, and a representative figure is shown."
NCBI gene IDs:
"HTT: 3064
SUMO-1: 7341"
Figure legend: "(A) Under limiting SUMO conditions (1/4 SUMO-1, lanes 3-9), PIAS1 increases HTT-SUMO modification above 1/4 SUMO alone. Purified HTT (arrow) and SUMO-HTT (boxed region) were detected using anti-HTT. Graph depicts quantitation of the Ni-NTA western blot using the Odyssey Infrared Imaging Software (LI-COR) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100."
NCBI gene IDs:
"HTT: 3064
PIAS1: 8554"
Figure legend: "(B) Western analysis of overexpression of HTTex1 (46QP-H4 or 3R), SUMO-2 (GFP-SUMO-2), and all the PIAS proteins (PIAS1, PIASxα, PIASxβ, PIAS3, and PIASy). Under nonlimiting SUMO-2 conditions, PIAS1 enhances SUMO modification of HTT. WC TCA shows overall myc-actin (transfection control) and HTT levels. Graph quantitating the ratio of HTT purified versus the HTT modified using the Odyssey (LICOR)."
NCBI gene IDs:
"HTT: 3064
PIAS1: 8554
PIASxα: 9063
PIASxβ: 9063
PIAS3: 10401
PIASy: 51588"
Figure legend: "(C) Left panel is the autoradiography results of a GST pull-down assay showing that radiolabeled human PIAS1 interacts with HTTex1p. Right panel is a phosphorimager analysis of GST pull-downs, performed in triplicate, showing the percentage of 35S-labeled PIAS protein that bound the GST proteins: GST alone, unexpanded HTT with and without the proline-rich region (20QP and 20Q), expanded HTT with and without the proline-rich region (51QP and 51Q), and the proline-rich region alone (Pro). Error bars were calculated as an estimate of SE = STDEV(n)/SQRT(n-1)."
NCBI gene IDs:
"HTT: 3064"
Figure legend: "(B) Longer HTT polypeptides are modified by SUMO-1. Western blot overexpressing HTTex1p (46QP-H4 or 3R), unexpanded HTT-586 fragment (25Q-586 or 25Q-3R-586 aa), and expanded HTT-586 fragment (137Q-586 or 137Q-3R-586 aa) with SUMO-1 (GFP-SUMO-1). Cell lysates were subjected to HTT immunoprecipitation (IP) using HTT antibody. Western analysis performed with the Odyssey (LI-COR) allows detection of HTT (data not shown) and SUMO simultaneously and shows that all forms of HTT are SUMO-1 modified using the anti-GFP antibody. HTTex1p is covalently SUMO-1 modified (lane 3), and the modification disappears when Lys are mutated to Arg (3R) (lane 4). Both unexpanded (lane 4 and 5) and expanded HTT-586 fragments (lane 6 and 7) are covalently SUMO-1 modified in both the presence and absence of the three Lys in the N-terminal region of HTT (3R). Free SUMO-1 is indicated with the arrow, and SUMO-modified HTT is indicated by the boxes. Inset on the right, from a replicate experiment, shows comigration of expanded HTT (anti-HTT) and SUMO-1 (anti-GFP) displayed in the gray scale and in color when the two antibodies are merged (HTT in red, SUMO-1 in green, and yellow when colocalizing)."
NCBI gene IDs:
"HTT: 3064
SUMO-1: 7341"
Figure legend: "(C) Bait plasmids (HTT-586-25Q or HTT-586-73Q aa) were transformed into the L40ccua MATa yeast strain. Yeast clones encoding bait proteins were individually mated against a matrix of MATα yeast clones encoding 16,888 prey proteins (with Gal4 activation domain fusions) using pipetting and spotting robots. Diploid yeasts were spotted onto SDIV (-Leu-Trp-Ura-His) agar plates for selection of PPIs as well as nylon membranes placed on SDIV agar plates for β-galactosidase assays. After 5-6 days of incubation at 30ºC, digitized images of the agar plates and nylon membranes were assessed for growth and β-galactosidase activity using the software Visual Grid (GPC Biotech)."
NCBI gene IDs:
"Gal4: "