EMBO/sd-character-level-ner huggingface dataset:
Figure 6Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets. Western blot analysis of precipitated MsrB2, Parkin and ubiquitinated MsrB2 (UB). Confocal microscopy in individual platelets in HC vs DM assessing for CoxIV (mitochondria marker), ubiquitin and MsrB2. Colocalization is demomstrated by arrows and quantitated. (colocalization ubiquitin, MsrB2; **p=0.0003 vs. HC). MsrB2 ubiquitination assay after transient transfection of MsrB2-GFP (1µg) with RFP empty vector (E.V.) or RFP-Parkin (3µg) in HEK293. After transfection (48hrs), an IP was performed using GFP-Trap bead, followed by Western blot analysis using UB and MsrB2 and Parkin antibodies. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
FAM83D & CK1α interact only in mitosis: A: Immunoblot analysis of wild type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cell lines.Figure 1A: Immunoblot analysis of wild type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cell lines. Data information: All blots are representative of at least 3 independent experiments. B: Proteomic analysis on asynchronous (AS), Nocodazole- or STLC-synchronised mitotic (M) FAM83DGFP/GFP knockin (KI) U2OS KI cells. The Venn diagram depicts the top proteins which were identified as FAM83D interactors in AS, M, or both AS and M conditions, in both nocodazole and STLC treatments (for a detailed analysis procedure, see the methods section). C: Schematic highlighting whether a mitotic interaction was previously known between FAM83D and the interacting proteins D: AS or nocodazole-synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations (IP). Extracts (input) and IP samples were analysed by immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. E: KI cells synchronised in mitosis with either nocodazole (M Noc.) or STLC (M STLC) were collected by shake-off, and drug-treated cells that remained adherent after shake-off (AS Noc.; AS STLC) were lysed and subjected to GFP TRAP IP. AS cells and free-GFP-expressing 2G-PAI1 U2OS cells were included as controls. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. F: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samples G, H: AS or nocodazole-synchronised (M) WT U2OS cells were subjected to IP with IgG and either anti-FAM83D-coupled sepharose beads (G), or anti-CK1α-coupled sepharose beads (H). Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. I: KI cells were synchronised in G2 with RO-3306, or arrested in mitosis (M) using STLC. STLC-treated shake-off cells were washed and re-plated, and cells lysed at the indicated time points after STLC wash-out. Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. J: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samples K: AS or STLC-synchronised (M) HeLa, A549 and HaCaT cells were subjected to IP with either IgG- or anti-FAM83D-coupled sepharose beads. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.
Figure 5(A) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) /anti-ZO-1 (red), anti-Ezrin-pT567 (green) /anti-ZO-1 (red), anti-Lamp1 (green) and anti-Sqstm1/p62 (green) antibodies from 6-month-old miR-211-/- sacrificed five months after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer. (B) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6-month-old miR-211-/- mice. Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1, Ezrin, Ezrin-pT567, Sqstm1/p62 and LC3. The plots show the quantification of the indicated proteins normalized to the β-Actin loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice). Mann and Whitney test (DMSO vs WT; NSC668394 vs DMSO), *p ≤ 0.05, ***p≤0.005. (C) Cathepsin B (CtsB) activity in RPE lysates from miR-211-/- and WT mice sacrificed 3h after light on at 10 AM (diurnal condition). RPE was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. CtsB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-), *p ≤ 0.05. (D) GUSB activity from retina from miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). Retina was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. GUSB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of GUSB activity ± s.e.m. of independent experiments (n=3 mice). Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-).
Figure 3A-D. Viability of primary human PMNs following incubation with a LD90 of LukED (78.12 nM), HlgAB (78.12 nM), HlgCB (39.1 nM), or LukSF-PV (19.53 nM) and the indicated dominant-negative toxins using the viability dye CellTiter. Open white symbols indicate no treatment. Filled black symbols indicate treatment with dominant-negative toxins. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 5 donors. E. Viability of primary human PMNs following incubation with filtered culture supernatants collected after 8 hours from CA-MRSA USA300 strain LAC using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 6 donors. F. Viability of primary human PMNs following incubation with 1.25% filtered culture supernatants and a dose response of the indicated dominant-negative (DN) toxins using the viability dye CellTiter. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors. G. Viability of primary human PMNs during a 3-hour ex vivo infection with the S. aureus Newman toxinless strain overproducing the indicated toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Bars indicate Mean ± SEM, with n = 3 donors. H. Viability of primary human PMNs during a 4-hr ex vivo infection with a MOI of 50 of S. aureus Newman toxinless strain overproducing the indicated toxins in the presence of a dose response of the DN toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors.
Figure 8A. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were immunostained for endogenous p62. Accumulation of p62 was quantified by counting p62 positive puncta per cell from 3 independent experiments (Mean ± SEM; unpaired t-test, **** P ≤ 0.0001); N (cells) = Ctrl: 74, C9orf72: 55). C9orf72 knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm. B. Primary cortical neurons (DIV5) were transduced with 4TU/cell EmGFP non-targeting control miRNA (Ctrl) or C9orf72 miRNA (green); for rescue experiments the cells were additionally transduced with 4TU/cell mVenus-tagged C9orf72s and C9orf72L (verified by immunoblot, Appendix Fig S2). Neurons were immunostained for endogenous p62 3 days post transduction. Accumulation of p62 was quantified by counting p62 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ** P ≤ 0.01; N (cells) = Ctrl miRNA: 80; C9orf72 miRNA: 80; C9orf72 miRNA+C9orf72L+C9orf72S: 75). Scale bar = 10 µm.
Figure 6(A) Schematic representation of S. cerevisiae Mdy2. Structural models of the N-terminal domain (NTD) (amino acids 1-59 of Mdy2, green, with Get4 in red, PDB ID: 2WPV), the central UbL domain (amino acids 74-148 of Mdy2, grey, PDB ID: 4GOC), the Cterminal dimerization domain (DD, amino acids 152-212 of Mdy2, blue, PDB ID: 2LNZ) of Mdy2. (B) In vitro degradation of Mdy2 proteins by purified S. cerevisiae proteasome. Mdy2 (red solid circles), Mdy2ΔN-95 (blue solid circles) and Mdy2-95 (green solid circles) were degraded. Removing the N-terminal domain of Mdy2 (Mdy2ΔN, black solid circles) or adding its binding partner Get4 (red open squares, dash line) or the proteasome-binding competitor UbLMdy2 (red open diamonds, dotted line) stabilized Mdy2Get4. When Mdy2 binding buried the N-terminal domain, addition of a 95 amino-acid tail derived from S. cerevisiae cytochrome b2 at the Cterminus restored degradation of Mdy2 (green open squares, dotted line). The graph plots the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m. (C) Degradation of Mdy2 in vivo. The steady-state accumulation of overexpressed C-terminally HA-tagged full-length Mdy2 and a mutant in which the N-terminal domain was deleted (Mdy2ΔN) in S. cerevisiae, in the absence of Get4, the presence of endogenously expressed Get4 or overexpressed Get4. Overexpressed Get4 was detected through an N-terminal Flag-tag. (D) Steady-state accumulation of endogenous Mdy2 in S. cerevisiae, with deleted (get4∆) or endogenous (wt) Get4 in the presence or absence of the proteasome inhibitor bortezomib. Protein levels were determined by western blotting for the HA-tag, Flag-tag, Mdy2 or Scs2 of SDS-PAGE gels of S. cerevisiae protein extracts. Protein loading levels in each lane were estimated in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.
Figure 2A. Immunoblot analysis of stable U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of WT or mutant forms of GFP-FAM111A. B. DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse-labeled with EdU and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image-based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). See also Appendix Figure S6A. C. Cells treated as in (B) were pre-extracted, fixed and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC (n>2000 cells per condition). D-F. Quantification of data in (C) (red bars, mean). Cells in S phase were identified based on EdU positivity. See also Appendix Figure S6B-D. G. Analysis of FAM111A interactors. U2OS/GFP-FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP) and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/-DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1). H. U2OS or U2OS/∆FAM111A cells were subjected to IP with IgG (control) or RFC1 antibody followed by immunoblotting with indicated antibodies. I. U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies. J. As in (C), except that cells were stained with RFC1 antibody (n>2000 cells per condition). K. Quantification of data in (J) for S phase (EdU-positive) cells (red bars, mean).
Figure 2A Confocal image of LimE in waves (scale bar, 20 μm). B Top, kymograph of LimE in a fixed region across which a piece of wave propagates, with white arrows pointing to the same puncta. Bottom, box plot of puncta's lifetime (n=100 puncta). C The coupled STEN-CEN model, with FS and RS (FC and RC) denoting the activator and inhibitor of STEN (CEN) respectively. The interaction between STEN and CEN is shown through the grey arrow. D A typical phase-plane trajectory of the state in STEN, where the initial inhibition level (r0) and threshold (th) are marked. The inset kymograph shows a typical STEN. E A typical trajectory of CEN in phase space showing threshold as in D. Although the nullclines of STEN and CEN are the same, the trajectory of CEN is significantly shorter resulting in smaller waves as seen from the inset kymograph. F A STEN-CEN organization profile with different instances in time marked for the corresponding phase plane trajectories in G. G (left) Plot showing the trajectory of the state in phase plane during the rising profile of STEN (i, from F). The rise of STEN shifts the CEN activator nullcline upwards creating a sharp rise in CEN activity (1, from F). (middle) The initial fall of STEN (ii, from F) resulting in a refractory zone of CEN as its nullcine is lowered (2, from F). (right) As the fall of STEN slows down, CEN reaches a firing position resulting in the second peak of CEN (3, from F). H A two-dimensional snapshot of a propagating wave (direction of propagation indicated with the dashed arrow). A line scan corresponding to the STEN-CEN profile is shown on the right. I Stochastic simulations of the model in c, showing the output from STEN (red, top right), CEN (green, top left) and the overlay (bottom right). A zoomed-in version of a CEN wave is shown corresponding to the white box in the CEN. Three line scans are plotted on the bottom right corresponding to the white lines in the overlay panel, showing the double-peak wave architecture of the STEN-CEN model.
Figure 2 Figure 2. Immunodominance of PbA-derived MHC II peptides during blood stage malaria induced after pRBC infection (A) Experimental scheme to analyze splenic CD4 T cell responses at day 6 following inoculation of PbA pRBC. (B) Gating strategy to calculate the proportion of CD11a+ CD49d+ activated CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC, OVA peptide (IR17) or ETRAMP10.2 peptide (NL17). (C) Percentage of IFN/TNF-double producing cells among activated CD4 T cells for each peptide. Basal level with MutuDC alone was subtracted. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates. (D) Hierarchy of immunodominance, depicted as the percentage of each peptide-specific response with respect to the total parasite-specific response obtained with PbA pRBC-loaded MutuDC. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates.
Figure 4A Y2H assays showing that SEU interacts with SDG4 and SDG25. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions. B Schematic diagram of the WOX5 and PCR amplicons (indicated as letters A-I) used for ChIP-qPCR. Data information: PCR amplicons are shown in (B). C SDG4 physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. ACT7, control. Data information Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (C) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test). D, E ChIP-qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter. ChIP signal was normalized to the WT. Individual values (black dots) are shown. Data information: Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (D), (E) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test). F, G Expression pattern of pWOX5::GFP in the indicated genotypes at 5 DAG. Scale bars: 20 µm. H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5::GFP. GFP signal intensity in the WT was set to 1. n denotes the total number of scored samples. Data information: In (H) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test). I RT-qPCR analysis of the relative expression levels of WOX5 in WT and ashr3-1 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Individual values (black dots) are shown. Data information: In (I) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test). J-M mPS-PI staining of stem cell niche in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indiate the CSCs. The numbers denote total number of scored samples, with same phenotypes showing in (J-M). Scale bars: 10 μm. N Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data information In (N), data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).
S.Figure 7.S. typhimurium-infected macrophages exhibited signs of autophagocytosis. Caspase-1−/− BMDPM were infected with wild-type S. typhimurium or it isogenic translocation-defective sipD mutant. 6 h after infection, macrophages were either stained with MDC to label autophagosomes (I) or processed for EM (II) as described in the legend to Fig. 3. Macrophages infected with wild-type S. typhimurium (but not those infected with the sipD mutant) exhibited a large number of vesicular structures that stained with MDC (I)  Under the electron microscope (II), macrophages infected with wild-type Salmonella exhibited multivesicular structures resembling autophagosomes (A-D) Often, electron-dense material, presumably damaged organelles, were readily observed within the multivesicular structures (B-D). In contrast, macrophages infected with type III secretion-defective sipD mutant did not exhibit the large number of multivesicular structures observed in wild-type-infected cells. Bars (A and E) 500 nm; (B, C, D and F) 250 nm.
Figure 5A t-SNE plots showing the original (left) and re-clustered (right) periosteal lineage cells by integrated analysis of homeostatic and stress conditions. Top 10 PCs were chosen for the clustering. B Violin plots showing the expression of feature genes for each cluster. C Pseudotime trajectories of 4 clusters. Cells were colored as in (A). D Dot plots showing the top5 GO terms enriched for each cluster. E Heatmap showing the AUC score of regulons enriched in each cluster. Z-score (column scaling) was calculated. Representative regulons were highlighted on the top. F Immunostaining of Sca-1 on femur sections of 8-week-old Lepr-Cre; tdTomato mice. Arrows indicated Sca-1+ tdTomato+ cells inside cortical bones of the femur diaphysis. Arrowheads indicated Sca-1+ tdTomato+ cells in the femur periosteum. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm. G Representative flow cytometry plots of long bone periosteum in 8-week-old Lepr-Cre; tdTomato mice. The percentage of DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1+/- cells were shown. H Representative CFU-F colony images. Two hundred DAPI- CD45- Ter119- Tie2- tdTomato+ (Lepr-Cre+), DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1+ (Lepr-Cre+Sca-1+) or DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1- (Lepr-Cre+ Sca-1-) cells were sorted from the limb bone periosteum of Lepr-Cre; tdTomato mice and cultured for 8 days, followed by crystal violet staining. I, J The average number of CFU-F colonies formed (I) and the average cell number per colony (J) were shown (n=4 independent experiments). The statistical significance of differences was assessed using one-way ANOVAs with Tukey's multiple comparison tests. All data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.
Figure 1Work flow to identify virus-induced small peptides in Arabidopsis. B. GUS activity of VISP1Pro::VISP1-GUS transgenic Arabidopsis plants infected by mock buffer or CMV-2blm at 7 dpi. Scale bar = 5 mm. C. Amino acid sequence alignment of AtVISP1 and its homologues from Arabidopsis lyrata (Al), Brassica rapa (Br), Brassica napus (Bn), Brassica oleracea (Bo), Brassica cretica (Bc), Arabis alpine (Aa), and Arabis nemorensis (An). The conserved ubiquitin-interacting motif (UIM) and Arginine/lysine-rich motif (ARM) are indicated by lines. D. BiFC analysis of interactions between VISP1 or VISP1mUIM and ATG8a or ATG8f in N. benthamiana leaves. Images were taken at 60 hpi. Scale bar = 20 μm. E. Co-IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM. N. benthamiana leaves were agroinfiltrated and treated with 2 mM 3-MA at 48 hpi, and then for IP with anti-Flag beads 12 h later. F. GST pull-down analysis of the interactions between ATG8a with VISP1 or VISP1mUIM.
Figure 1A Schematic diagram of HOIP orthologues in Drosophila melanogaster (D. melanogaster) and Homo sapiens (H. sapiens). D. melanogaster CG11321 is predicted to contain B-box (blue), Npl4 zinc finger (NZF) (navy), ubiquitin (Ub)-associated (UBA1 and UBA2) (pink), and a C-terminal catalytic region, RING between RING (RBR)-C. The RBR-C region consists of RING1 (R1; red), In Between RING (IBR; yellow), RING2 (R2; light green) and Linear Ub chain Determining Domain (LDD; green). PNGase/Ub-associated (PUB) (sky blue) and Zinc finger (ZF) (lavender) domains were found in H. sapiens but not in D. melanogaster. B Ub chain interaction of the LUBEL-NZF and UBA2 domains. Immobilized GST-NZF or GST-UBA2 was incubated with Lys (K) 48-, K 63-linked, or linear di-Ub (Ub2) chains and bound Ub chains were subjected to immunoblotting using anti-Ub antibody or a linear linkage-specific Ub antibody (anti-linear Ub antibody). Loading of GST-fusion proteins was visualized by Ponceau S staining.