EMBO/SourceData huggingface dataset:
Figure 6Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). (n = 9 mice [Emx1-cWT] and 9 mice [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).Decreased delta power (normalized to total power) in NREM, but not WAKE, oscillations in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). Note also the normal theta power (normalized to total power) in REM oscillations in Emx1-Cre;Ptprdfl/fl mice. (n = 9 [Emx1-cWT] and 9 [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).(D) Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period and decreased REM duration in the first half of the light-on period in Ptprd-meA-/- mice (2.5-4 months). Note also that Ptprd-meA-/- mice also display increased WAKE duration and decreased NREM duration during the light-off period. (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).Decreased delta power (normalized to total power) in NREM (both light-off and light-on periods) and WAKE (light-on period) oscillations, and decreased theta power in REM (mainly light-on period) oscillations in Ptprd-meA-/- mice (2.5-4 months). (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).
Figure 1WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Lethality was followed-up. LPS-treated mice (n=5) are depicted as black circles and DEX/LPS-treated mice (n=5) are depicted as white circles. P-values for survival curves were analyzed with a Fisher's exact test.WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Blood samples were taken 2 hours after LPS and TNF levels were measured in serum (n=4-5).WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Three hours after LPS challenge, dextran labeled with FITC was administered by oral gavage. Five hours after gavaging, blood plasma was collected and fluorescence was measured as a degree of intestinal permeability (λexc/λem = 488/520 nm) (PBS: n=3, LPS and DEX/LPS: n=5).RNA sequencing (RNAseq) results of IECs of WT mice (n=3) treated with PBS or 12.5 mg/kg LPS for 6 hours. Heatmap with selection of LPS-induced genes. Color key shows the log2 of normalized counts.WT mice (n=5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated and gene expression was measured via qPCR.
Figure 2WT mice (n=3-5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated 6 hours after LPS challenge. (A) p-MLKL and MLKL protein levels and (B) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control. Expression levels were quantified using FIJI and normalized to ACTIN levels. P-values were calculated using 1-way ANOVA.Gene expression (Defa6, lysozyme) was measured via qPCR. P-values were calculated using 1-way ANOVA. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.H&E staining was performed on ileum samples. Scale bar: 50µm.Lysozyme expression (in red) was detected via IHC, with Hoechst counterstaining. Scale bars: 50µm. Quantification of lysozyme expression. For every picture (n=3-4 per treatment), a selection of 10 crypts was made. Lysozyme positive signal was quantified in this region of interest (ROI) using an automated script in QuPath. Values are the positive area relative to total area of ROI.
Figure 3TNFR1+/+ (n=9) and TNFR1-/- mice (n=11) were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TLR4fl/fl mice (n=7) and TLR4VillinKO (n=9) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TNFR1fl/fl mice (n=13) and TNFR1VillinKO (n=10) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TNFR1+/+ and TNFR1-/- mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.TNFR1fl/fl and TNFR1VillinKO mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.
Figure 4GRfl/fl (n=8,) and GRLysMKO mice (n=4) were treated with 2.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.GRLysMKO mice were treated with PBS (n=12) or 10 mg/kg DEX (n=13) before a lethal dose of LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TNF measurement in the serum of GRfl/fl (n=4) and GRLysMKO mice (n=3). Mice were treated with 2.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.TNF measurement in the serum of GRfl/fl and GRLysMKO (n=4). Mice were treated with PBS or a lethal dose of LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) , with or without pre-treatment with 10 mg/kg DEX and blood was drawn 2 hours later. TNF concentration was measured with ELISA.GRfl/fl (n=4) and GRLysMKO mice (n=3-4) were treated with 2.5 mg/kg LPS. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.GRfl/fl and GRLysMKO mice (n=4) were treated with a PBS or LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) with or without 10 mg/kg DEX pre-treatment of 30 minutes. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.
Figure 5GRfl/fl (n=8) and GRVillinKO mice (n=10) were treated with 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.GRVillinKO mice were treated with PBS (n=9) or 10 mg/kg DEX (n=10) before 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n= 4). Mice were treated with 5 mg/kg LPS and blood was drawn 2 hours later.TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n=4). Mice were treated with PBS or LPS (12.5 and 5 mg/kg for GRfl/fl and GRVillinKO mice respectively) with or without pre-treatment with 10 mg/kg DEX for 30 minutes. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.GRVillinKO mice were treated with PBS (n=8) or a neutralizing P55 (TNFR1) antibody (n=9) 2 hours before and 8 hours after 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.Ileum was isolated from non-stimulated GRfl/fl (n=4) and GRVillinKO mice (n=4) and gene expression was measured via qPCR. Expression levels are shown as fold induction with GRfl/fl expression values set at 1. P-values were calculated using a student's t-test.GRfl/fl (n=4) and GRVillinKO mice (n=4) were treated with 5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR.
Figure 6Left: LPS dose-response curves of GRwt/wt (n=3-7 per LPS dose) and GRdim/dim mice (n=3-7 per LPS dose) treated with 1.5 mg/kg LPS. Right: LD50 values of LPS are depicted on top of each bar for each group.GRdim/dim mice were treated with PBS (n=5) or 10 mg/kg DEX (n=5) before 1.5 mg/kg LPS and survival was monitored. P-values were analyzed with a Fisher's exact test.TNF measurement in the serum of GRwt/wt (n=5) and GRdim/dim mice (n= 5). Mice were treated with 1.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.TNF measurement in the serum of GRwt/wt and GRdim/dim mice (n=4) treated with PBS or LPS (12.5 and 1.5 mg/kg in GRwt/wt and GRdim/dim mice respectively) with or without pretreatment with 10 mg/kg DEX. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.GRdim/dim mice were treated with PBS (n=6) or a neutralizing P55 (TNFR1) antibody (n=8) 2 hours before and 8 hours after 1.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.Tofacitinib (n=6) or vehicle (n=7) was given orally to GRdim/dim mice. After 1.5 mg/kg LPS injection survival was monitored and p-values were analyzed with a Fisher's exact test.GRwt/wt (n=4) and GRdim/dim (n=4) mice were treated with 1.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.GRdim/dim mice (n=4) were treated with PBS or 1.5 mg/kg LPS with or without 10 mg/kg DEX pretreatment. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.
Figure 3For SPR measurements, IL-27Rα or GP130 were immobilized on the chip surface by biotin-streptavidin interaction and IL-27 were flowed across the chip in solution. SPR data are representative of three biological replicates. Kinetic charts for IL-27Rα (A) and GP130 (B). Concentrations used are shown on the curves. Data traces were fitted using a 1:1 interaction model (black) to quantify the kinetics (kon, koff) and binding affinity (KD) of the interactions.For SPR measurements, IL-27Rα were immobilized on the chip surface by biotin-streptavidin interaction and p28 were flowed across the chip in solution. SPR data are representative of three biological replicates. (C, Equilibrium chart for p28 binding to IL-27Rα (C, left panel) and curve-fitting to data points generated at various concentrations of p28 (C, right panel).For SPR measurements, GP130 were immobilized on the chip surface by biotin-streptavidin interaction and p28 were flowed across the chip in solution. SPR data are representative of three biological replicates. (D) Equilibrium chart for p28 binding GP130.(F,G) Dose response curves for pSTAT1 (F) and pSTAT3 (G) in resting mouse CD8+ T cells. Cells were stimulated with mIL-27 or p28 for 15 minutes with the indicated doses. Data shown are the mean of four biological replicates with error bars depicting standard error of the mean.
Figure 1(B-D) Flow cytometric quantification of DHE>Eth. positive wild type yeast cells (allowing the detection of both dead cells and cells exhibiting sub-lethal oxidative stress) after 42 h (B) or 66 h (C, D) expressing Abeta constructs. Dot plots show all data points along with the mean (bar) ± s.d.. n = 5-12 biologically independent cultures. ***, p < 0.001, **, p < 0.01, *, p < 0.05. ANOVA with Tukey's post-hoc test.(E) Annexin V (AV)/PI co-staining to assess cell death in yeast expressing A42 after 48 h. Data represent means ± s.d. n = 6 biologically independent cultures. ***, p < 0.001 (comparing PI positive populations). Unpaired, two-tailed t-test.(F) Immunoblot of whole cell extract (WCE) of wild type yeast cells after 16 h expression of Abeta constructs. For western blot (WB) Abeta-specific antibody (Abeta) 6E10 was used showing long (Long exp.) and short time exposure (Short exp.). Ssc1 was used as a loading control.(G) Confocal microscopy of wild type yeast cells after 18 h of Abeta expression. Colors indicate fluorescence intensity.
Figure 2(A) Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial, microsomal and cytosolic fractions of wild type yeast cells after 18 h expression of EGFP-Abeta42 (A42), EGFP-Abeta40 (A40) and EGFP empty vector (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Purity of fractions was tested with antibodies against Cyc1 (mitochondria), Sec61 (microsomes) and Pgk1 (cytosol).(B) Cellular ATP content of wild type yeast cells after 42 h expression of EGFP-A42 (A42) or EGFP empty vector (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 16 biologically independent cultures. **, p < 0.01. Unpaired, two-tailed t-test.(C) Relative protein abundance of EGFP-Abeta42 (A42) versus empty vector (ev) expressing wild type yeast cells. The significant subset of cytosolic heat shock proteins (HSP) detected in a proteomics analysis of isolated mitochondria is depicted. Log2 SILAC ratios of A42/ev of two independent proteome measurements are shown. Dot plots show all data points along with the mean (line). Significance was determined using an outlier test (Significance A, p <0.003).(D) Screen of 123 deletion strains assessing Abeta42 (A42)-induced toxicity (assessed by DHE>Eth. positive cells indicative of the sum of sub-lethal oxidative stress and cell death) in yeast. Relative A42-toxicity normalized to wild type (WT) cells is depicted. Potential hits (mutants that reduce A42 toxicity) are shown in green. Data represent means ± s.d.. n ≥ 3 biologically independent cultures.
Figure 3Quantification of cells positive for PI indicating cell death at indicated time points after start of expression of EGFP-A42 (A42) or EGFP empty vector (ev) of wild type (WT) (A) and YDJ1 deleted (Δydj1) cells (B). Mean ± s.d.. n = 5 biologically independent cultures. Comparisons by two-way ANOVA (mixed-design) followed by simple main effects (***, p < 0.001, vs. control).Immunoblot of whole cell extract (Input) and eluate of FLAG-tagged Ydj1. Immunoprecipitation (IP: FLAG) of YDJ1 deletion strain (Δydj1) cells expressing EGFP-A42 (A42) or EGFP empty vector (ev) and co-overexpressing Ydj1-FLAG using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).Confocal microscopy of wild type (WT) yeast cells expressing EGFP-A42 (A42) only and co-overexpressing Ydj1-FLAG or with the corresponding vector control after 18 h of expression. Colors indicate fluorescence intensity.Confocal microscopy of wild type (WT) yeast cells expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG after 18 h of expression. Mitochondria were visualized with MitoTracker Red (magenta). See also Fig EV2J, EV1C.Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.) and cytosolic (Cyt.) fractions of wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Tom22-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.Ratio of full length EGFP-A42 in the mitochondrial fraction / cytosolic fraction, using densitometry quantification of the immunoblot representatively shown in Fig 3F and Appendix Fig S1C from three/4 independent experiments. Dot plots show all data points along with the mean (line/bar) ± s.d.. n = 3-4 biologically independent cultures. *, p < 0.05. Unpaired, two-tailed t-test.Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity was measured at two time points (t0 and t2, 2 h after CH administration) and normalized to t0 in wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 8 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc. See also Appendix Fig S2D.Quantification of the ratio between EGFP-A42 tetramer and monomer in wild type (WT) and YDJ1 deletion strain (Δydj1) expressing EGFP-A42 (J) as well as between WT expressing EGFP-A42 only and co-overexpressing Ydj1-FLAG (K) at indicated time points. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8(J) n=12(K) biologically independent cultures. ***, p < 0.001; **, p < 0.01; *, p < 0.05. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2A-B.
Figure 4Quantification of propidium iodide (PI)-staining positive cells indicating cell death at indicated time points of expression of EGFP-A42 (A42) Δydj1 cells with co-expression of DnaJA1-FLAG (DnaJA1) (B) or corresponding empty vector controls (ev) (A). Mean ± SD n = 4-6 biologically independent cultures. Comparisons by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus control).(C)Immunoblot (WB) of whole-cell extract (Input) and eluate of FLAG-tagged DnaJA1 immunoprecipitation (IP: FLAG) of wild-type strain (WT) expressing EGFP-A42 (A42) and co-expressing DnaJA1-FLAG (A1) using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity of YDJ1 deletion strain (Δydj1) and wild type (WT) cells expressing EGFP-A42 (A42) only or co-expressing DnaJA1 was measured at two time points (t0 and t2) and normalized to t0. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2C.Immunoblot (WB) of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.), and cytosolic (Cyt.) fractions of YDJ1-deleted cells (Δydj1) after 18 h expression of EGFP-A42 (A42) only or co-expressing DnaJA1-FLAG (DnaJA1) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time (Short exp.) exposure (two sections from one immunoblot). Cox4-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 (A1) and quantified by densitometry in (G).Quantification of the oligomer-to-monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in (F). Dot plots show all data points along with the mean (line) ± SD n = 3 biologically independent cultures. P-values by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus A42 control). See also Appendix Fig S2D.A42 beta-sheet-rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ. Data represent means of at least eight measurements. See also Appendix Fig S1E.
Figure 5(A) Immunoblot of cytosol-enriched cerebral tissue homogenate (Input) and eluate of Abeta42 or DnaJA1 using Abeta-specific antibody (6E10) and DnaJA1-specific antibody (DnaJA1), respectively, showing optimal and long exposure (Long exp.). Immunoprecipitation using magnetic beads without antibody, DnaJA1 or 6E10 antibody of female (15 months old) wild type (WT) or 3xTg (PS1M146V/APPSwe/tauP301L) mice. Sections showing monomer and low-n oligomers, dodecamer and full-length amyloid precursor protein (APP) are from 1 immunoblot.(B) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). PonceauS serves as a loading control. (C) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). Representative immunoblot is shown in (B). Dot plots show all data points along with the mean (bar) ± s.d.. n = 4-5 independent patients. *, p < 0.05. Unpaired, two-tailed t-test.
Figure 6(A) Representative confocal microscopy of 10-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).(B) Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 18-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).(C-D) Average intensity (C) and total area (D) of Abeta (6E10) signal from confocal images representatively shown in (A) from 12 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 10-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 12. *, p < 0.05. Unpaired, two-tailed t-test.(E-F) qPCR analysis of Droj2-mRNA levels of 3-6 days old female (E) and male (F) flies expressing human Abeta42 (UAS-A42) of w1118 wild type flies (Droj2+/+) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression (Droj2+/+ ctrl). Reference gene is Rpl32. Dot plots show all data points along with the mean (line) ± s.d.. n = 3. **, p < 0.01. One sample t-test against 1.(G) Immunoprecipitation (IP: 6E10) of w1118 wild type flies (Droj2+/+) using synthetic Abeta42 added to the fly head extract (Input). Abeta-specific antibody 6E10 was used for Abeta42 and DnaJA1-specific antibody for Droj2 immunoblot detection. Showing input, supernatant, supernatant after washing (WS) and eluate.
Figure 7(A-B) Survival of female (A) and male (B) w1118 wild type flies (Droj2+/+) and Droj2 knockdown flies (Droj2+/-) with expression of human Abeta42 (UAS-A42) or control flies without expression (ctrl), upon supplementation of sugar (10% sucrose) with 20 mM MnCl2. Survival was determined at indicated time points. n = 6 with 100-120 flies per experiment. The indicated p-value refers to the interaction (int.) term of a Cox Proportional Hazards model comparing Abeta42 toxicity (UAS-A42 vs. ctrl) and Droj2 expression (Droj2+/+ vs. Droj2+/-) as main factors. The following pairwise comparisons of the indicated groups survival were done by Log Rank test (****, p < 0.0001; ns, p > 0.05).(C) Aversive associative memory performance 2 min after training of aged (18 days old) male Droj2 knockdown flies (Droj2+/-) and corresponding isogenic w1118 wild type flies (Droj2+/+) both expressing human Abeta42 (UAS-A42) of six independent biological replicates. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6. *, p < 0.05. Unpaired, two-tailed t-test.(D) Representative confocal and gSTED deconvolved images of Kenyon cells in 15-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A-specific antibody (ATP5A, green) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).(E-F) Solidity (E) and circularity (F) of ATP5A-stained mitochondria from fly brain gSTED deconvolved images representatively shown in (D) from 8-10 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 15-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8-10. *, p < 0.05. Unpaired, two-tailed t-test.
Figure 1SUMO conjugation assays performed under multiple turnover conditions using wild-type or mutant forms of RPA, SUMO and Siz2. Only the 80-minute time points are presented to highlight the formation of Rfa1-SUMO and Siz2-SUMO species. The complete time series are presented in Appendix Fig S1. Bands for Mw marker are fully annotated in Figure 1B.
Figure 2SUMO conjugation assays performed under multiple turnover conditions using Siz2∆CT and different RPA-DNA complexes. SDS-PAGE gels were stained with Coomassie. Bands for Mw marker are fully annotated in Figure 1B.
Figure 3Plot showing the initial rate of SUMO conjugation versus RPA-DNA concentration as obtained from single-turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA-DNA complexes. Representative gels are shown in Appendix Fig S4.
Figure 4SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2∆CT lacking (A) or including (B) its SAP domain. In both cases, reactions were done in technical triplicate and SDS-PAGE gels were stained with SYPRO. Only one representative gel is shown. Bands for Mw marker are fully annotated in Figure 1B.