Journal: Journal of Circadian Rhythms

Article Title: Circadian Oscillation Pattern of Endoplasmic Reticulum Quality Control (ERQC) Components in Human Embryonic Kidney HEK293 Cells

doi: 10.5334/jcr.219

Figure Lengend Snippet: The members of the retrotranslocation and dislocation components of ERQC exhibit a rhythmical expression pattern. a. mRNA expression levels of p97/VCP, Ufd1, Npl4, SVIP and Derlin1 in the HEK293 cells across a 48 h circadian cycle. The total RNAs were isolated from the HEK293 cells collected every 4 h during a 48 h circadian period and cDNAs were synthesized. Genes of interest were amplified by PCR and visualized on agarose gels. b. Relative gene expression quantities corresponding to three biological replicates were collected every 4 h (blue circles). The intensity of the bands was analyzed densitometrically and normalized to TBP expression. Fold changes in mRNA expression levels were determined by comparison to the expression level at 0 h. Oscillation (represented as a continuous curve) was modeled, via curve fitting analysis. A nonlinear curve fitting analysis was conducted, which fitted a sinusoid function [A*sin(Bt + c)] to the data including the replicates. Genes showing an R 2 correlation greater than 0.8 in non-linear curve fitting analyzes were kept. (n = 3).

Article Snippet: Rabbit polyclonal antibodies p97/VCP (10736-1-AP)(1:10000), gp78 (16675-1-AP)(1:3000) and Bmal1 (14268-1-AP)(1:2500) were obtained from Proteintech, rabbit monoclonal antibody against Hrd1 (#14773)(1:3000) was provided from Cell Signaling Technology.

Techniques: Expressing, Isolation, Synthesized, Amplification, Gene Expression, Comparison

Journal: Journal of Circadian Rhythms

Article Title: Circadian Oscillation Pattern of Endoplasmic Reticulum Quality Control (ERQC) Components in Human Embryonic Kidney HEK293 Cells

doi: 10.5334/jcr.219

Figure Lengend Snippet: Hrd1, gp78 and p97/VCP expression exhibit circadian rhythmicity in HEK293 cells. a. The rhythmic protein accumulation levels of Hrd1, gp78, p97/VCP and Bmal1 in HEK293 cells. Samples were collected every 4 h in a 32 h circadian period. Beta-actin was used as a loading control. b. The intensity of the protein bands was densitometrically analyzed and data represent means ±SEM in the graph (n = 3) * p < 0.05.

Article Snippet: Rabbit polyclonal antibodies p97/VCP (10736-1-AP)(1:10000), gp78 (16675-1-AP)(1:3000) and Bmal1 (14268-1-AP)(1:2500) were obtained from Proteintech, rabbit monoclonal antibody against Hrd1 (#14773)(1:3000) was provided from Cell Signaling Technology.

Techniques: Expressing, Control

Journal: BMC Medical Genomics

Article Title: Construction and verification of a prognostic model for prostate cancer based on ribosome biogenesis-related genes

doi: 10.1186/s12920-025-02262-w

Figure Lengend Snippet: Validation of the expression of prognostic genes. a Expression of prognostic genes in TCGA-PRAD. b PCR results of NVL. c PCR results of FBLL1. d PCR results of SMARCA4. e PCR results of RRS1. f HPA (Human Protein Atlas) results of NVL. g HPA results of FBLL1. h HPA results of SMARCA4. i HPA results of RRS1. j WB results of genes

Article Snippet: After rinsing with TBS (Signalway Antibody, USA) several times and blocking with 5% non-fat milk (BBI, China), the membranes were incubated with anti- SMARCA4 primary antibody (1:2000;21634-1-AP; Proteintech; USA), anti- FBLL1 primary antibody (1:1000;PA5-49014;Invitrogen; USA), anti- RRS1 primary antibody (1:500;153291-AP, Proteintech, USA) and anti- NVL primary antibody (1:3000;16970-1-AP; Proteintech; USA) overnight.

Techniques: Biomarker Discovery, Expressing

Journal: Molecular Biology of the Cell

Article Title: VPS13D interacts with VCP/p97 and negatively regulates ER- mitochondrial interactions

doi: 10.1091/mbc.e21-03-0097

Figure Lengend Snippet: FIGURE 3: VPS13D interacts with p97, is required for p97 stability. (A) The domain organization of VPS13D and two truncation mutants. (B) GFP-trap assays indicated that the UBA and VAB domains independently interacted with p97. (C) Western blots showed the effects of p97 overexpression on GFP-VAPB (WT or disease mutant P56S and T46I). (D) Quantification of Western blots results . Three independent assays were performed. Ordinary one-way ANOVA, followed by Turkey’s multiple comparisons test. Mean ± SD. (E) Western blots showed that VPS13D suppression decreased the level of endogenous p97 in U2OS and HEK293 cells. (F) Quantification of Western blots results in (E). Two-tailed unpaired Student’s t test. Mean ± SD. (G) GFP-trap assays indicated that VPS13D suppression resulted in a reduced level of SNAP-VAPB coimmunoprecipitated with p97-GFP. (H) Western blots of cycloheximide chase assays showing the changes of the level of p97-GFP after CHX treatment. (I) Normalized ratio of p97-GFP to tubulin in

Article Snippet: mCherry-mito7 (55102), mitoBFP (49151), p97-GFP (23971), pRK5HA-ubiquitin-WT (17608), pRK5-HA-ubiquitin-K48 (17605), and pRK5-HA-ubiquitin-K63 (17606) were purchased from Addgene. eBFP-peroxisome was previously described (Ji et al., 2017).

Techniques: Western Blot, Over Expression, Mutagenesis, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: VPS13D interacts with VCP/p97 and negatively regulates ER- mitochondrial interactions

doi: 10.1091/mbc.e21-03-0097

Figure Lengend Snippet: FIGURE 4: VPS13D suppression results in defects of mitochondrial motility, morphology, cellular distribution, and mtDNA synthesis. (A) Left: whole cell image of U2OS expressing ER (green) and mitochondrial (red) markers were treated either with scrambled or VPS13D siRNA. Right: overlays of mitochondrial dynamics time course with two insets. Colors depict time points in the sequence, with white indicating relative immobility (Supplementary Video 1). (B) Mitochondrial perimeter in scrambled siRNA (669 mitochondria from 52 cells), VPS13D siRNAs–treated U2OS cells (754 mitochondria from 53 cells), VPS13D/VAPB/PTPIP51 siRNAs–treated U2OS cells (742 mitochondria from 51 cells), and VPS13D siRNAs/p97-GFP–treated U2OS cells (746 mitochondria from 49 cells). One-way ANOVA followed by Tukey’s multiple comparisons test. Mean ± SD. (C) Mitochondrial localization in scrambled siRNA (102 cells), VPS13D siRNAs–treated U2OS cells (150 cells), VPS13D/VAPB/PTPIP51 siRNAs–treated U2OS cells (95 cells), and VPS13D siRNAs/p97-GFP–treated U2OS cells (92 cells). Chi-squared and Fisher’s exact tests. (D) Confocal images of live polG2-GFP stable U2OS cells expressing mCherry-mito7 (red) up treatments with scrambled (Top) or VPS13D siRNAs (Bottom). Yellow arrows indicate replicating mtDNA at mitochondrial tips in scrambled cells; blue arrows indicate

Article Snippet: mCherry-mito7 (55102), mitoBFP (49151), p97-GFP (23971), pRK5HA-ubiquitin-WT (17608), pRK5-HA-ubiquitin-K48 (17605), and pRK5-HA-ubiquitin-K63 (17606) were purchased from Addgene. eBFP-peroxisome was previously described (Ji et al., 2017).

Techniques: Expressing, Sequencing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma

doi: 10.1186/s13046-021-02220-1

Figure Lengend Snippet: AHSA1 promotes MM proliferation and BTZ resistance through activating CDK6 and PSMD2 respectively. A-C Using Co-IP assay followed by MS, CDK6 and PSMD2 were selected among candidate genes of the proliferation-related and drug-resistance genes in ARP1 WT, AHSA1-OE cells, AHSA1-OE cells treated with Bufalin or BTZ, respectively. D WB analysis showed the expressions of CDK6 and PSMD2 in AHSA1-OE (Left) and AHSA1-KD cells (Right). E Co-IP experiment further confirmed the interaction between HSP90 with CDK6 and PSMD2 in ARP1 and H929 cells using HSP90 antibody as bait. F Co-IP experiment showed that CDK6 directly interacted with HSP90 in ARP1 and H929 cells. G Co-IP assay confirmed that PSMD2 interacted with HSP90 in ARP1 and H929 cells. H Proteasome activity assay showed that overexpression of AHSA1 in ARP1 and H929 cells resulting in high proteasome activity. I Validation of PSMD2 overexpression in ARP1 and H929 PSMD2-OE cells relative to WT cells. J Proteasome activity assay showed that overexpression of PSMD2 in ARP1 and H929 cells led to high proteasome activity. K-L Effects of BTZ on the cell viability of ARP1 ( K ) and H929 ( L ) cells with or without PSMD2 overexpression. The data are expressed as mean ± SD.* p< 0.05 , **p <0.01, *** p <0.001

Article Snippet: Antibodies were as follows: AHSA1 (83036, Abcam, UK); HSP90 (13171-1-AP, ProteinTech Group, China); PSMD2 (14748-1-AP, ProteinTech Group, China); CDK6 (14052-1-AP, ProteinTech Group, China); HA (51064-2-AP, ProteinTech Group, China); MYC (16286-1-AP, ProteinTech Group, China); FLAG (F-4020, Merck KGaA, Germany); GAPDH (60004-1-Ig, ProteinTech Group, China); PARP (9542S, Cell Signaling Technology, USA); Caspase-3 (9662S, Cell Signaling Technology, USA); β-actin (4970S, Cell Signaling Technology, USA); Rabbit IgG (a7016, Beyotime Institute of Biotechnology, China) and mouse IgG (a7028, Beyotime Institute of Biotechnology, China).

Techniques: Co-Immunoprecipitation Assay, Activity Assay, Over Expression, Biomarker Discovery

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma

doi: 10.1186/s13046-021-02220-1

Figure Lengend Snippet: Bufalin decreases cellular proliferation and PI resistance induced by AHSA1/HSP90 in MM cells. A-B Effects of Bufalin (60nM) and BTZ (10nM) incubation for 48h on cell apoptosis of ARP1 ( A ) and H929 ( B ) WT and AHSA1-OE cells. C The rate of drug-induced apoptosis was shown in the histogram. D Effects of Bufalin on the expression of CDK6 and PSMD2 in ARP1 and H929 WT and AHSA1-OE cells. E-F Co-IP assay revealed that Bufalin interfered the interaction between HSP90 and AHSA1 in ARP1 and H929 cells. G Co-IP assay confirmed the interaction between AHSA1, HSP90, CDK6, PSMD2 and the activated form of CDK6, phosphorylation of Y13 site at CDK6. H-J Proteasome activity assay verified that Bufalin inhibited proteasome activity in ( H ) ANBL6 WT/DR cells, I ARP1 and H929 AHSA1 WT/OE cells and J PSMD2 WT/OE cells. The data are expressed as mean ± SD.* p<0.05, **p <0.01, *** p <0.001

Article Snippet: Antibodies were as follows: AHSA1 (83036, Abcam, UK); HSP90 (13171-1-AP, ProteinTech Group, China); PSMD2 (14748-1-AP, ProteinTech Group, China); CDK6 (14052-1-AP, ProteinTech Group, China); HA (51064-2-AP, ProteinTech Group, China); MYC (16286-1-AP, ProteinTech Group, China); FLAG (F-4020, Merck KGaA, Germany); GAPDH (60004-1-Ig, ProteinTech Group, China); PARP (9542S, Cell Signaling Technology, USA); Caspase-3 (9662S, Cell Signaling Technology, USA); β-actin (4970S, Cell Signaling Technology, USA); Rabbit IgG (a7016, Beyotime Institute of Biotechnology, China) and mouse IgG (a7028, Beyotime Institute of Biotechnology, China).

Techniques: Incubation, Expressing, Co-Immunoprecipitation Assay, Phospho-proteomics, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma

doi: 10.1186/s13046-021-02220-1

Figure Lengend Snippet: AHSA1-K137 is identified as the site-specific targeting of Bufalin and KU-177. A hHsp90α-hAHSA1 complex model. a Overview of hHsp90α (left) interacted with hAHSA1 (right, colored in yellow). Both hHsp90α and hAHSA1 were represented in colored cartoon and corresponding transparent surface. The N-terminal (15-287), linker (288-323), middle domain (324-476), and C-terminal (477-698) domain of hHsp90α were colored in cyan, red, blue and green, respectively. b Detailed information of hHsp90α binding with hAHSA1. The key residues corresponding to hAHSA1 bound with hHsp90α were shown as orange and blue sticks, respectively. The key interactions between residues were depicted by red dotted lines. B Predicted binding modes of Bufalin targeting hAHSA1. Bufalin was shown in red sticks, and hAHSA1 was shown in yellow cartoon. Key residues were shown in gray sticks, and hydrogen bonds were depicted by dotted lines. Phenylalanine in mutation of AHSA1-N131F was shown in magenta sphere. C MST results of Bufalin on wild and site-directed mutagenesis (SDM) of hAHSA1. AHSA1-K137 was involved in hydrogen bond interactions with Bufalin, and this hydrogen bond interaction disappeared with mutation to alanine. D-E AHSA1-K137 was involved in hydrogen bond interactions with its co-chaperone, HSP90, and AHSA1/HSP90 client protein, CDK6 and PSMD2, and this hydrogen bond interaction disappeared while mutation to alanine or double mutation K137A/N131F, as demonstrated by Co-IP using HA antibody as bait in ARP1 HSP90-OE cells followed by WB. AHSA1-OE plasmid was linked with HA tag, while HSP90-OE plasmid was linked with FLAG tag. F The chemical structure of KU-177. G Structural details of the predicted binding modes of KU-177. KU-177 was shown in green sticks, and key residues of hHsp90α (green cartoon) and AHSA1 (yellow cartoon) were shown in orange and gray sticks, respectively. Conformational changes from a hHsp90α-binding conformation (blue transparent cartoon) to a KU-177 “Induced Fit” conformation (yellow cartoon), as well as corresponding residues, K137 and N131, were also presented. H Predicted binding modes of KU-177 targeting hAHSA1. KU-177 was shown in green sticks, and hAHSA1 was shown in yellow cartoon. Key residues were shown in gray sticks, and hydrogen bonds were depicted by dotted lines. Phenylalanine in mutation of N131F was indicated in magenta sphere. I MST results of KU-177 on wild and site-directed mutagenesis (SDM) of hAHSA1. K137 was involved in hydrogen bond interactions with KU-177, and this hydrogen bond interaction would disappear with mutation to alanine

Article Snippet: Antibodies were as follows: AHSA1 (83036, Abcam, UK); HSP90 (13171-1-AP, ProteinTech Group, China); PSMD2 (14748-1-AP, ProteinTech Group, China); CDK6 (14052-1-AP, ProteinTech Group, China); HA (51064-2-AP, ProteinTech Group, China); MYC (16286-1-AP, ProteinTech Group, China); FLAG (F-4020, Merck KGaA, Germany); GAPDH (60004-1-Ig, ProteinTech Group, China); PARP (9542S, Cell Signaling Technology, USA); Caspase-3 (9662S, Cell Signaling Technology, USA); β-actin (4970S, Cell Signaling Technology, USA); Rabbit IgG (a7016, Beyotime Institute of Biotechnology, China) and mouse IgG (a7028, Beyotime Institute of Biotechnology, China).

Techniques: Binding Assay, Mutagenesis, Co-Immunoprecipitation Assay, Plasmid Preparation, FLAG-tag

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma

doi: 10.1186/s13046-021-02220-1

Figure Lengend Snippet: KU-177 decreases MM cell proliferation and PI resistance induced by AHSA1/HSP90 in vitro . A-B Effects of 48 h treatment with KU-177 on cell viability of ARP1 ( A ) and H929 ( B ) WT and AHSA1-OE cells. C-E Effects of 48 h treatment with KU-177 (50 μM) on cell apoptosis of ARP1 ( C ) and H929 ( D ) WT and AHSA1-OE cells. F Effects of 48 h treatment with KU-177 (30 μM) on cell viability of flow MRD-positive peripheral cells from first diagnosed and relapsed MM patients. H-K Co-IP assay revealed that 48 h treatment with KU-177 (30 μM) inhibited the interaction between HSP90 and AHSA1 in AHSA1-OE cells. L-N Proteasome activity assay showed that 48 h treatment of KU-177 (30 μM) inhibited proteasome activity in AHSA1 WT/OE cells ( L ), PSMD2 WT/OE cells ( M ) and ANBL6 WT/DR ( N ) cells. The data are expressed as mean ± SD.* p<0.05, **p <0.01, *** p <0.001. The data are expressed as mean ± SD.* p<0.05, **p <0.01, *** p <0.001

Article Snippet: Antibodies were as follows: AHSA1 (83036, Abcam, UK); HSP90 (13171-1-AP, ProteinTech Group, China); PSMD2 (14748-1-AP, ProteinTech Group, China); CDK6 (14052-1-AP, ProteinTech Group, China); HA (51064-2-AP, ProteinTech Group, China); MYC (16286-1-AP, ProteinTech Group, China); FLAG (F-4020, Merck KGaA, Germany); GAPDH (60004-1-Ig, ProteinTech Group, China); PARP (9542S, Cell Signaling Technology, USA); Caspase-3 (9662S, Cell Signaling Technology, USA); β-actin (4970S, Cell Signaling Technology, USA); Rabbit IgG (a7016, Beyotime Institute of Biotechnology, China) and mouse IgG (a7028, Beyotime Institute of Biotechnology, China).

Techniques: In Vitro, Co-Immunoprecipitation Assay, Activity Assay

Journal: EMBO Reports

Article Title: UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation

doi: 10.1038/s44319-023-00027-z

Figure Lengend Snippet: ( A ) Immunoblot of BiP and ATF4 expression levels in HFT wildtype cells treated with 1 µM thapsigargin (Tg), 5 µM of the ATP-competitive p97 inhibitor CB-5083, or both for the indicated timepoints (0–6 hours). ( B , C ) Band intensity quantifications of BiP ( B ) and ATF4 ( C ) corresponding to Figure EV1A reporting the fold change compared to the untreated condition ( n = three biologically independent samples). ( D ) Immunoblot of HFT wildtype cells depleted of BAG6 with siRNA for 48 hours before treatment with 1.5 mM DTT for the indicated timepoints (0–8 hours). ( E ) Band intensity quantifications of BiP and ATF4 corresponding to Figure EV1D at the 8-hour timepoint. ( n = four biologically independent experiments). ( F ) Transcript levels of xbp1s and total xbp1 in HEK-293T wildtype and UBXN1 KO cells quantified by quantitative real-time PCR. Cells were treated with 10 nM thapsigargin (Tg) for 4 hours as indicated. ( n = three biologically independent samples). ( G ) Immunoblot of BiP and ATF4 expression in cells depleted of UBXN1 with siRNA. Cells were treated with 1.5 mM DTT for the indicated time points. ( H ) Band intensity quantifications of BiP and ATF4 from Figure EV1G at the 8-hour time point. ( n = four biologically independent experiments). ( I ) Immunoblot of ATF6 activation in cells depleted of UBXN1 with siRNA. Cells were treated with 1.5 mM DTT and 1 µM Bortezomib (BTZ). ( J ) ATF6 activation was measured by band intensity quantification and calculation of the percentage of cleaved ATF6 to total ATF6. The ratio of the percentage of ATF6 activation in UBXN1 KO cells to wildtype is reported. ( n = three biologically independent samples). Data information: Data are means ± SEM (*, **, ***, **** where P < 0.05, 0.01, 0.001, and 0.0001, respectively.) One-way ANOVA with Dunnetts multiple comparisons test ( B , C ). Unpaired two-tailed t test ( E , H , J ). One-way ANOVA with Tukey’s multiple comparisons test ( F ).

Article Snippet: The rabbit UBXN1 (16135-1-AP 1:7000 dilution), p97 (10736-1-AP), BAG6 (26417-1-AP), ATF6 (24169-1-AP), Sec61β (51020-2-AP 1:3000 dilution), TIMM17A (11189-1-AP), Aconitase 2 (11134-1-AP), calnexin (10427-2-AP), CYC1 (10242-1-AP), and G3BP1 (13057-2-AP) antibodies were obtained from Proteintech Inc; rabbit BiP (3177 dilution), peIF2α (3398 1:500 dilution), and MCM3 (4012S) antibodies were obtained from Cell Signaling Technology; mouse PCNA (sc-56 1:3000 dilution), ATF4 (sc-390063 1:500 dilution), TOMM20 (sc-17764), TOMM70 (sc-390545), TIMM23 (sc-514463), ubiquitin (P4D1; sc8017), β-actin (sc-69879 1:3000 dilution), GAPDH (sc-47724 1:3000 dilution), c-myc (sc-40 1:3000 dilution), Sec61α (sc-393182 1:500 dilution), and TRAPα (sc-373916) were from Santa Cruz Biotechnology; mouse ubiquitin FK2 (04-263 1:100 dilution) for immunofluorescence and Anti-Puromycin, clone 12D10 (MABE343 1:3000 dilution) was from EMD Millipore; mouse FLAG-M2 antibody (F3165 1:1000 dilution) was from Sigma Aldrich.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Activation Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation

doi: 10.1038/s44319-023-00027-z

Figure Lengend Snippet: ( A ) Immunoblot of puromycin incorporation into HFT wild-type and UBXN1 KO cells. Cells were pulsed with 1 µM puromycin or 1 µM puromycin in combination with 10 µg/mL cycloheximide (CHX) for 30 min. When applicable, cells were pretreated with 1.5 mM DTT for 1 h before 1 µM puromycin pulse for 30 min. The level of puromycin incorporation reflects the rate of protein synthesis. ( B ) Puromycin, DTT, and CHX plots correspond to the intensity of each lane in the immunoblot in A . The intensity of the traces for each wild-type and UBXN1 KO sample was determined by the plot profile feature in Fiji. The x -axis represents the distance along the lane. ( C ) Myc-tagged GFP, wildtype, UBX domain mutant, or UBA domain mutant UBXN1 was expressed in cells for 48 hours before 1 µM puromycin pulse for 30 minutes. ( D ) Quantification of each lane intensity from C . ( n = three biologically independent samples). ( E ) Polysome profile traces of HEK-293T wild-type and UBXN1 KO cells. Cells were treated with 2 mM DTT for 60 minutes where indicated. The 40S, 60S, and 80S ribosomal subunits are labeled as well as actively translating polysomes. Traces to the right correspond to the polysomes seen in the main trace. ( F ) Polysome profile traces of HEK-293T cells depleted of p97 with siRNA. Cells were treated with 2 mM DTT for 60 min where indicated. The 40S, 60S, and 80S ribosomal subunits are labeled as well as actively translating polysomes. Traces to the right correspond to the polysomes seen in the main trace. Data information: Data are means ± SEM (** where P < 0.01) One-way ANOVA with Tukey’s multiple comparisons test ( D ). .

Article Snippet: The rabbit UBXN1 (16135-1-AP 1:7000 dilution), p97 (10736-1-AP), BAG6 (26417-1-AP), ATF6 (24169-1-AP), Sec61β (51020-2-AP 1:3000 dilution), TIMM17A (11189-1-AP), Aconitase 2 (11134-1-AP), calnexin (10427-2-AP), CYC1 (10242-1-AP), and G3BP1 (13057-2-AP) antibodies were obtained from Proteintech Inc; rabbit BiP (3177 dilution), peIF2α (3398 1:500 dilution), and MCM3 (4012S) antibodies were obtained from Cell Signaling Technology; mouse PCNA (sc-56 1:3000 dilution), ATF4 (sc-390063 1:500 dilution), TOMM20 (sc-17764), TOMM70 (sc-390545), TIMM23 (sc-514463), ubiquitin (P4D1; sc8017), β-actin (sc-69879 1:3000 dilution), GAPDH (sc-47724 1:3000 dilution), c-myc (sc-40 1:3000 dilution), Sec61α (sc-393182 1:500 dilution), and TRAPα (sc-373916) were from Santa Cruz Biotechnology; mouse ubiquitin FK2 (04-263 1:100 dilution) for immunofluorescence and Anti-Puromycin, clone 12D10 (MABE343 1:3000 dilution) was from EMD Millipore; mouse FLAG-M2 antibody (F3165 1:1000 dilution) was from Sigma Aldrich.

Techniques: Western Blot, Mutagenesis, Labeling

Journal: EMBO Reports

Article Title: UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation

doi: 10.1038/s44319-023-00027-z

Figure Lengend Snippet: ( A ) Densitometry quantifications of the entire lanes corresponding to Fig. ( n ≥ three biologically independent experiments). ( B ) Immunoblot of HFT wildtype and UBXN1 KO cells pulsed with 1 µM puromycin for 30 min after pre-treatment with 1.5 μM thapsigargin for 30 minutes where indicated. ( C ) Immunoblot of 1 µM puromycin incorporation into control cells, or cells depleted of p97 or UBXN1 with siRNA for 48 hours. Cells were pre-treated with 1 µM thapsigargin for 30 minutes where indicated. ( D ) Quantification of the whole lane corresponding to each lane in the immunoblot in Figure EV5C ( n = four biologically independent samples). ( E ) Immunoblot validating the siRNA knockdown corresponding to Fig. . ( F ) Immunoblot of ribosome and polysome fractions collected with corresponding UV traces showing 40S, 60S, 80S and polysome fractions. RPL4 is a marker for the 60S subunit and RPS3 is a marker for the 40S subunit. Whole cell lysates (WCL) are shown for input. ( G ) UBXN1 KO cells have similar viability to wildtype cells under resting conditions. Data shown in Main Fig. is shown here without normalization to untreated in each genotype. We observe no differences in viability between wildtype and UBXN1 KO cells in untreated conditions allowing for this normalization. Fold change of the fluorescence measured by fluorescence-based cytotoxicity assay. Cells were treated with 1.5 μM thapsigargin in combination with 10 μg/ml cycloheximide where indicated. Values were normalized to wildtype untreated. ( n = three biologically independent samples). Data information: Data are means ± SEM (*, **, *** and **** where P < 0.05, 0.01, 0.001 and 0.0001, respectively.) Unpaired two-tailed t test ( A ). One-way ANOVA with Tukey’s multiple comparisons test ( D , G ).

Article Snippet: The rabbit UBXN1 (16135-1-AP 1:7000 dilution), p97 (10736-1-AP), BAG6 (26417-1-AP), ATF6 (24169-1-AP), Sec61β (51020-2-AP 1:3000 dilution), TIMM17A (11189-1-AP), Aconitase 2 (11134-1-AP), calnexin (10427-2-AP), CYC1 (10242-1-AP), and G3BP1 (13057-2-AP) antibodies were obtained from Proteintech Inc; rabbit BiP (3177 dilution), peIF2α (3398 1:500 dilution), and MCM3 (4012S) antibodies were obtained from Cell Signaling Technology; mouse PCNA (sc-56 1:3000 dilution), ATF4 (sc-390063 1:500 dilution), TOMM20 (sc-17764), TOMM70 (sc-390545), TIMM23 (sc-514463), ubiquitin (P4D1; sc8017), β-actin (sc-69879 1:3000 dilution), GAPDH (sc-47724 1:3000 dilution), c-myc (sc-40 1:3000 dilution), Sec61α (sc-393182 1:500 dilution), and TRAPα (sc-373916) were from Santa Cruz Biotechnology; mouse ubiquitin FK2 (04-263 1:100 dilution) for immunofluorescence and Anti-Puromycin, clone 12D10 (MABE343 1:3000 dilution) was from EMD Millipore; mouse FLAG-M2 antibody (F3165 1:1000 dilution) was from Sigma Aldrich.

Techniques: Western Blot, Control, Knockdown, Marker, Fluorescence, Cytotoxicity Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation

doi: 10.1038/s44319-023-00027-z

Figure Lengend Snippet: ( A ) Relative xbp1s expression quantified by quantitative real-time PCR. Cells were treated with 10 nM thapsigargin in combination with 10 μg/ml cycloheximide where indicated. ( n = three biologically independent samples. ( B ) Fold change of the fluorescence measured by fluorescence-based cytotoxicity assay. Cells were treated with 1.5 μM thapsigargin in combination with 10 μg/ml cycloheximide where indicated. Values were normalized to untreated for each genotype. Note we observe no differences in viability between wild-type and UBXN1 KO cells in untreated conditions allowing for this normalization ( n = three biologically independent samples. ( C ) Model Figure. UBXN1 plays an important role in ER-quality control as a repressor of translation and the UPR. Loss of UBXN1 increases protein synthesis. Increased abundance of ER proteins leads to protein misfolding and activation of the UPR which reduces cell viability. Some p97 ERAD clients may require UBXN1 for degradation. Data information: Data are means ± SEM (*, **, ***, **** where P < 0.05, 0.01, 0.001, and 0.0001, respectively.) One-way ANOVA with Tukey’s multiple comparisons test ( A , B ). .

Article Snippet: The rabbit UBXN1 (16135-1-AP 1:7000 dilution), p97 (10736-1-AP), BAG6 (26417-1-AP), ATF6 (24169-1-AP), Sec61β (51020-2-AP 1:3000 dilution), TIMM17A (11189-1-AP), Aconitase 2 (11134-1-AP), calnexin (10427-2-AP), CYC1 (10242-1-AP), and G3BP1 (13057-2-AP) antibodies were obtained from Proteintech Inc; rabbit BiP (3177 dilution), peIF2α (3398 1:500 dilution), and MCM3 (4012S) antibodies were obtained from Cell Signaling Technology; mouse PCNA (sc-56 1:3000 dilution), ATF4 (sc-390063 1:500 dilution), TOMM20 (sc-17764), TOMM70 (sc-390545), TIMM23 (sc-514463), ubiquitin (P4D1; sc8017), β-actin (sc-69879 1:3000 dilution), GAPDH (sc-47724 1:3000 dilution), c-myc (sc-40 1:3000 dilution), Sec61α (sc-393182 1:500 dilution), and TRAPα (sc-373916) were from Santa Cruz Biotechnology; mouse ubiquitin FK2 (04-263 1:100 dilution) for immunofluorescence and Anti-Puromycin, clone 12D10 (MABE343 1:3000 dilution) was from EMD Millipore; mouse FLAG-M2 antibody (F3165 1:1000 dilution) was from Sigma Aldrich.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Fluorescence, Cytotoxicity Assay, Control, Activation Assay

Journal: Chinese Science Bulletin

Article Title: A proteome-wide screen identifies valosin-containing protein as an essential regulator of podocyte endoplasmic reticulum stress

doi: 10.1007/s11434-012-5250-8

Figure Lengend Snippet: Figure 4 Verification of VCP and HSPa5 in LC-MS/MS by real-time PCR and Western blotting. (a) The degree of change of VCP and HSPa5 on days 14 and 21 in the pHN model detected by LC-MS/MS. (b) Real-time PCR determined the mRNA expression level of VCP and HSPa5; at days 14 and 21, the two proteins showed increased expression, and significant differences were observed between days 14 and 21 compared to normal tissue. *P < 0.05, n = 3. (c) Western blotting determined the protein expression level of VCP and HSPa5; at days 14 and 21, the two proteins showed increased expression, and signifi- cant differences were observed between days 14 and 21 compared to normal tissue. *P < 0.05, n = 3.

Article Snippet: Reagents used were acetonitrile (AN; Fisher Scientific, Waltham, MA, USA), formic acid (FA; Fisher Scientific), dithiothreitol (DTT; Sigma, St. Louis, MO, USA), iodoacetemide (IAA; Amresco, Solon, OH, USA), NH4HCO3 (Sigma), trypsin enzymolysis liquid (Promega, Madison, WI, USA), BCA protein concentration detection reagent kit (Pierce, Rockford, IL, USA), tunicamycin (Tm; Sigma), rabbit polyclonal anti-HSPa5, VCP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-rabbit IgG (Santa Cruz Biotechnology).

Techniques: Liquid Chromatography with Mass Spectroscopy, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: Chinese Science Bulletin

Article Title: A proteome-wide screen identifies valosin-containing protein as an essential regulator of podocyte endoplasmic reticulum stress

doi: 10.1007/s11434-012-5250-8

Figure Lengend Snippet: Figure 5 VCP was inhibited by RNAi, and ERS was induced by tunicamycin (Tm). MPCs were pretreated with 10 g/mL Tm for 10 h to induce ERS; the expression levels of VCP and HSPa5 were both upregulated, and significant differences were observed compared with the control. *P < 0.05, n = 3. MPCs were transfected with siRNA and VCP protein expression was inhibited (†P < 0.05, n = 3), but HSPa5 expression did not change. Cells were transfected with siRNA of VCP and treated with Tm; HSPa5 expression increased, and a significant difference was observed between the Tm and Tm+siRNA groups (‡P < 0.05, n = 3), although VCP expression was considerable in the control.

Article Snippet: Reagents used were acetonitrile (AN; Fisher Scientific, Waltham, MA, USA), formic acid (FA; Fisher Scientific), dithiothreitol (DTT; Sigma, St. Louis, MO, USA), iodoacetemide (IAA; Amresco, Solon, OH, USA), NH4HCO3 (Sigma), trypsin enzymolysis liquid (Promega, Madison, WI, USA), BCA protein concentration detection reagent kit (Pierce, Rockford, IL, USA), tunicamycin (Tm; Sigma), rabbit polyclonal anti-HSPa5, VCP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-rabbit IgG (Santa Cruz Biotechnology).

Techniques: Expressing, Control, Transfection

Journal: Frontiers in Pharmacology

Article Title: Dissecting gastric cancer heterogeneity and exploring therapeutic strategies using bulk and single-cell transcriptomic analysis and experimental validation of tumor microenvironment and metabolic interplay

doi: 10.3389/fphar.2024.1355269

Figure Lengend Snippet: Identification and validation of M2 macrophage-related risk genes. (A) Violin plot of abundance of 22 subtypes of immune cells in risk subgroups. (B) Spearman’s correlation between infiltration of 22 subtypes of immune cells and individual TME-Met PI genes (n = 15) in TCGA STAD cohort. p values are shown as: * p < 0.05; ** p < 0.01; *** p < 0.001. (C) UMAP plots and (D) Violin plots showing expression patterns of CPVL, KYNU, CD36, and GPX3 in single-cell gastric cancer dataset (GSE112302). (E) UMAP plots showing expression patterns of CPVL, KYNU, CD36, and GPX3 in GSE167297 dataset. (F) Representative images of expression (brown, cell cytoplasmic/nucleus stain) and ( G ) IHC quantification of expression level of CPVL, KYNU, CD36, and GPX3 and marker of M2 macrophage (CD163) in the clinical samples of stomach adenocarcinoma (n = 8). ( H ) Pearson’s correlation of expression level of CPVL, KYNU, CD36, and GPX3 and marker of M2 macrophage (CD163) in the clinical samples of stomach adenocarcinoma.

Article Snippet: Following the washing step with 10% phosphate-buffered saline (PBS), the sections were blocked using 5% BSA and then exposed to primary antibodies against the following targets: KYNU (Proteintech, #11796-1-AP, Rabbit, 1:100), CPVL (Proteintech, #12548-1-AP, Rabbit, 1:200), CD36 (Proteintech, #18836-1-AP, Rabbit, 1:800), GPX3 (Signalway Antibody, #27854, Rabbit, 1:800), and CD163 (Cell Signaling, #93498S, Rabbit, 1:250).

Techniques: Biomarker Discovery, Expressing, Staining, Marker

Journal: Frontiers in Pharmacology

Article Title: Dissecting gastric cancer heterogeneity and exploring therapeutic strategies using bulk and single-cell transcriptomic analysis and experimental validation of tumor microenvironment and metabolic interplay

doi: 10.3389/fphar.2024.1355269

Figure Lengend Snippet: Overview of the risk stratification of gastric cancer patients based on tumor microenvironment and metabolism interplay. Extensive analysis revealed 15 risk genes expressed by diverse TME components. M2 macrophages upregulated KYNU, GPX3, CPVL, and CD36. Fibroblasts expressed VCAN, ANGPT2, LOX, GPX3, SNCG, GFRA1, and NOX4, impacting proliferation, mesenchymal transition, angiogenesis, and ROS production. PDE1B and CARD11 were associated with B and T lymphocytes, while PNMA2, KIT, and MAGEA3 were expressed by various gastric gland cells, such as chief cells, gland mucous cells, and proliferative cells. The TME-Met Interplay upregulated ECM biosynthesis and fatty acid metabolism. The high-risk subgroup showed resistance to immunotherapy and chemotherapy but responded to three molecular targeted drugs. Conversely, low-risk patients exhibited enriched glycolysis, glucose, and amino acid metabolism, with lower ECM content and sensitivity to immunotherapy and chemotherapy.

Article Snippet: Following the washing step with 10% phosphate-buffered saline (PBS), the sections were blocked using 5% BSA and then exposed to primary antibodies against the following targets: KYNU (Proteintech, #11796-1-AP, Rabbit, 1:100), CPVL (Proteintech, #12548-1-AP, Rabbit, 1:200), CD36 (Proteintech, #18836-1-AP, Rabbit, 1:800), GPX3 (Signalway Antibody, #27854, Rabbit, 1:800), and CD163 (Cell Signaling, #93498S, Rabbit, 1:250).

Techniques:

Journal: PLoS ONE

Article Title: A Ubiquitin Independent Degradation Pathway Utilized by a Hepatitis B Virus Envelope Protein to Limit Antigen Presentation

doi: 10.1371/journal.pone.0024477

Figure Lengend Snippet: ( A ) In order to test the hypothesis that ubiquitination is not important for the targeting of W/T M protein for proteasome dependent degradation, K141 and K160 on wild type M (MS) was mutated to Alanine (MSKA). The constructs were transfected into 293T cells and either left untreated (-) or treated with proteasome inhibitor epoxomicin (1.5 uM) for 16 h. The cells were then lysed and the intracellular M protein detected via immunoblot using a PreS2 antibody (a.a.13–26 of PreS2). The locations of the three species of wild type M protein are indicated. The asterisk is used to highlight the p30, which accumulate with proteasome inhibition. The level of β-actin was used to monitor loading and is shown below the M immunoblot. (B) The p30, gp33 and gp36 M proteins are ERAD substrates. MS (left) or MSKA (right) were either co-transfected into 293T cells with an empty vector, or a dominant negative P97 (P97QQ) expression vector and the level of M protein (for Pre-S2 domain), p97 and actin measured by immunoblot. Anti-PreS2 immunoblot shows that inhibition of functional P97 by over expression of dominant negative P97 rescued all of the transfected contructs, indicating that p97 is required for the degradation of the HBV M protein in a ubiquitin indepedent manner. (C) Removal of lysine residues indeed blocks ubiquitination of W/T M protein. HEK293T cells were either transfected with HA-Ub alone or cotransfected with MS or MSKA as indicated and left untreated or treated with the proteasome inhibitor Lactacystin (20 uM) for 16 hours. Anti-HA and anti-PreS2 immunoblots were used confirm the expression of each indicated protein. Co-immunoprecipitation was performed using HA affinity gel followed by western blot analysis with anti-PreS2 antibody to detect interaction between HA-tagged ubiquitin and wild type or mutant M proteins. Regardless of the presence of lysine residues (compare MS and MSKA), HA-tagged ubiquitin could not be detected on wild type M protein, confirming the lack of ubiquitin conjugation.

Article Snippet: Rabbit polyclonal anti-VCP (P97) and anti-OS-9 antibodies were purchased from Novus Biologicals, LLC (Littleton, CO).

Techniques: Ubiquitin Proteomics, Construct, Transfection, Western Blot, Inhibition, Plasmid Preparation, Dominant Negative Mutation, Expressing, Functional Assay, Over Expression, Immunoprecipitation, Mutagenesis, Conjugation Assay

Journal: PLoS ONE

Article Title: A Ubiquitin Independent Degradation Pathway Utilized by a Hepatitis B Virus Envelope Protein to Limit Antigen Presentation

doi: 10.1371/journal.pone.0024477

Figure Lengend Snippet: ( A ) Cys-48, Cys-65 and Cys-69 on wild type M protein was converted to Alanines, and the construct was named as CA. In order to study the role of ubiquitination in the rapid degradation of the CA mutant protein, all the lysine residues (K141, K160) were mutated to Alanines, and the construct was named as CAKA. Wild type M construct (MS), CA and CAKA were transfected into 293T cells and either left untreated (-) or treated with epoxomicin for 16 hours one day post-transfection. The accumulation of CA mutant protein in the presence of the proteasome inhibitor implies that this protein is being degraded in a proteasome dependent manner; however this degradation process does not require ubiquitination as removal of Lysines did not prevent the rapid degradation by the proteasome. The level of β-actin was used to monitor loading and shown below the M immunoblot (B) The p30, gp33 and gp36 CA M proteins are ERAD substrates. CA and CAKA were either co-transfected into 293T cells with an empty vector, or a dominant negative P97 (P97QQ) expression vector and the level of M protein (for Pre-S2 domain), p97 and actin measured by immunoblot. Anti-PreS2 immunoblot shows that inhibition of functional P97 by over expression of dominant negative P97 rescued all of the transfected contructs, indicating that p97 is required for the degradation of the HBV M protein in a ubiquitin indepedent manner. (C) Removal of lysine residues indeed blocks ubiquitination of CA. HEK293T cells were either transfected with HA-Ub alone or cotransfected with CA or CAKA as indicated, and either left untreated or treated with the proteasome inhibitor Lactacystin (20 uM) for 16 hours. As in , anti-HA and anti-PreS2 immunoblots were used to confirm the expression of each indicated protein. However, when lysine residues were removed (CAKA), none of the ubiquitinated misfolded M could be detected as there is no any specific signal comparing to the control lane in which HA-Ub alone was transfected in the cells, indicating that removal of Lysine residues indeed prevents ubiquitination of CA mutant proteins.

Article Snippet: Rabbit polyclonal anti-VCP (P97) and anti-OS-9 antibodies were purchased from Novus Biologicals, LLC (Littleton, CO).

Techniques: Construct, Ubiquitin Proteomics, Mutagenesis, Transfection, Western Blot, Plasmid Preparation, Dominant Negative Mutation, Expressing, Inhibition, Functional Assay, Over Expression, Control

Journal: Nature Structural & Molecular Biology

Article Title: VCP/p97-associated proteins are binders and debranching enzymes of K48–K63-branched ubiquitin chains

doi: 10.1038/s41594-024-01354-y

Figure Lengend Snippet: a , Sankey diagram connecting the six distance clusters to annotation clusters of DAVID gene ontology analysis colored by annotation enrichment score. b , Table of molecular functions and known binding motifs of proteins specifically associated with K48–K63-branched Ub chains (clusters 3 and 4). c , Schematic of p97 subcomplexes with varying Ub chain-binding preferences. d , Silver-stained SDS–PAGE analysis of HALO pulldown with recombinant HALO-tagged RFC1 UBD [190–246] and branched/unbranched Ub 4 containing K48 and K63 linkages.

Article Snippet: Antibodies were sourced from the indicated manufacturers and used at 1:2,000 dilution unless otherwise stated: anti-GFP (Abcam, ab290), anti-GFP (Proteintech, 50430-2-AP; 1:5,000), anti-VCP/p97 (Proteintech, 10736-1-AP; 1:4,000), anti-ATXN3 (Proteintech, 13505-1-AP), anti-Ub (Biolegend, P4D1), anti-Ub K48-specific (Sigma, Apu2), anti-UBE2N (Invitrogen, 37-1100), anti-α-tubulin (CST, 3837; 1:5,000) and anti-GAPDH (Proteintech, 10494-1-AP; 1:5,000).

Techniques: Binding Assay, Staining, SDS Page, Recombinant

Journal: Nature Structural & Molecular Biology

Article Title: VCP/p97-associated proteins are binders and debranching enzymes of K48–K63-branched ubiquitin chains

doi: 10.1038/s41594-024-01354-y

Figure Lengend Snippet: a – c , Pulldowns from U2OS cell lysates using agarose-immobilized NbSL3.3Q and subsequent western blot analysis of input and elution fractions with indicated antibodies. Cells were treated with DMSO, MG-132, NMS-873 or CB-5083 for 4 h. The quantification shows the total Ub enrichment in eluted protein relative to DMSO-treated samples ( n = 4 technical replicates; n = 3 for CB-5083; error bars denote the s.d. and the bar denotes the mean) ( a ). Cells were treated with NMS-873, CB-5083 or MG-132 for indicated time. Western blot analysis of total Ub, p97 and ATXN3 ( b ). Cells were treated with nonspecific siRNA or siRNA targeting ATXN3 for 48 h, supplemented with DMSO or NMS-873 (5 µM) treatment for 4 h before harvest ( c ). d , Quantification of c and additional replicates showing the total Ub in input and eluted protein fractions relative to control siRNA + DMSO samples ( n = 6 individual data points with the line showing the mean value ± s.d.). Indicated P values were determined by two-way ANOVA with Dunnett’s test. e , DUB assay using Miy2 (K48-specific) or AMSH (K63-specific), USP2 (unspecific) and ATXN3 (K63-specific, preference for K48–K63 branches) incubated for 1 h at 37 °C with Ub chains captured by anti-GFP pulldown from NbSL3.3Q-GFP-expressing U2OS Flp-In Trex cells (lanes 1–12) following treatment with DMSO (lanes 1–6) or NMS-873 (lanes 7–12) or recombinant K48–K63-branched Ub 3 chains (lanes 13–18). Samples were analyzed by western blotting for total Ub and K48-linked Ub and with Ponceau S for total protein. f , Representative live-cell images of recruitment UV microirradiation assay with U2OS cells stably expressing NbSL3.3Q-GFP, NbSL18-GFP (negative control) or GFP-DDB2 (positive control). NbSL3.3Q-GFP cells were treated either with control siRNA or UBE2N siRNA. Cells were imaged before damage and over a time course of 10 min following insult by 405-nm UV laser microirradiation at 9 J m − 2 . Nuclei are indicated in white and laser-targeted subnuclear locations are indicated in purple (GFP-DDB2, n = 79 cells; NbSl18-GFP, n = 76 cells; NbSL3.3Q-GFP, control siRNA, n = 144 cells; NbSL3.3Q-GFP, siUBE2N, n = 128 cells). Scale bars, 5 µm. g , Quantification of recruitment assay f represented as the average mean GFP intensity ± s.e.m. within the targeted subnuclear spot per nucleus. h , Representative live-cell images of retention UV microirradiation assay with U2OS cells stably expressing NbSL3.3Q-GFP treated with NMS-873 and either control or ATXN3 siRNA. Cells were subjected to localized laser microirradiation and subsequently followed for 1 h. Nuclei are indicated in white and laser-targeted subnuclear locations are indicated in purple (untreated, control siRNA, n = 29 cells; untreated, siATXN3, n = 20 cells; p97i, control siRNA, n = 52 cells; p97i, siATXN3, n = 26 cells). Scale bars, 5 µm. i , Kinetics of the half-times of recruitment and removal of NbSL3.3Q-GFP from sites of localized laser microirradiation were calculated from the time courses of individual cells of the retention assay ( h ). Data are shown as bars representing the mean GFP intensity half-times ± s.e.m. (*** P < 0.001 and **** P < 0.0001, determined by a Welch’s unpaired t -test). White circles indicate individual data points (two outliers are excluded from visualization for clarity). j , Quantification of maximum cumulative recruitment of NbSL3.3Q-GFP in retention assay ( h ). Data points represent the mean GFP intensity of individual cells ± s.e.m. k , Speculative mechanistic model for the role of K63 branches on K48-Ub chains during substrate processing by p97. K48–K63-branched chains may act as a priority signal for p97 through p97-associated branched-chain-binding adaptors. The presence of Ub branches on the distal end of the Ub chain favors threading of the proximal Ub and substrate through p97 for unfolding, while the distal Ub escapes the central pore. The K63-specific debranching activity of p97-associated DUB ATXN3 subsequently edits the branched chain of the processed substrate to a K48-linked Ub chain for proteasomal degradation.

Article Snippet: Antibodies were sourced from the indicated manufacturers and used at 1:2,000 dilution unless otherwise stated: anti-GFP (Abcam, ab290), anti-GFP (Proteintech, 50430-2-AP; 1:5,000), anti-VCP/p97 (Proteintech, 10736-1-AP; 1:4,000), anti-ATXN3 (Proteintech, 13505-1-AP), anti-Ub (Biolegend, P4D1), anti-Ub K48-specific (Sigma, Apu2), anti-UBE2N (Invitrogen, 37-1100), anti-α-tubulin (CST, 3837; 1:5,000) and anti-GAPDH (Proteintech, 10494-1-AP; 1:5,000).

Techniques: Western Blot, Control, Incubation, Expressing, Recombinant, Stable Transfection, Negative Control, Positive Control, Binding Assay, Activity Assay

Journal: Nature Structural & Molecular Biology

Article Title: VCP/p97-associated proteins are binders and debranching enzymes of K48–K63-branched ubiquitin chains

doi: 10.1038/s41594-024-01354-y

Figure Lengend Snippet: a) Pulldowns using NbSL3.3Q-immobilized agarose from U2OS cells treated with indicated inhibitors (NMS-873 − 5 μM, MG-132 − 10 μM, VER-155008 − 5 μM, tunicamycin − 5 μg/ml). Western blot analysis of total ubiquitin in input lysate and eluted proteins. b) U2OS cells treated with NMS-873, CB-5083 or MG132 and non-specific siRNA or siRNA targeting p97 or ATXN3 or both in combination. c) HEK293 Flp-In Trex cells were treated with tetracycline to induce expression of Ub G76V -GFP followed by p97-inhibition using NMS-873 (5 µM) for 4 hours. Subsequent pulldowns with NbSL3.3Q-immoblised agarose were analyzed by western blotting for total Ub and GFP. Note: The abcam anti-GFP antibody used here produces a non-specific band at approximately the same size as native GFP. d) Anti-GFP pulldown from U2OS cells expressing either NbSL3.3Q-GFP or NbSL18-GFP following p97-inhibition with NMS-873 analyzed by western blotting for total Ub and GFP. e) Western blot analysis of anti-GFP pulldowns from U2OS Flp-In Trex following tetracycline-induced expression of GFP, NbSL3.3Q-GFP, NbSL18-GFP or GFP-DDB2 visualized with anti-GFP antibody. f , g) Western blot analyses of U2OS Flp-In Trex used in UV micro-irradiation assays (Main Fig. ) following tetracycline-induced expression of NbSL3.3Q-GFP, NbSL18-GFP or GFP-DDB2 and treatment with NMS-873 or siRNA (non-targeting control, UBE2N or ATXN3). h) Representative images from timelapses of U2OS cells stably expressing NbSL3.3Q-GFP, pre-damage and 10 minutes after local 405 nm laser micro-irradiation at indicated laser intensities. Positions of damage events (purple) and nuclei (white) are highlighted. White scale bar is 5 µm. i) Quantification of NbSL3.3Q-GFP recruitment over 10 minutes at various laser intensities (assay in Fig. ). NbSL3.3Q-GFP recruitment is represented as the average mean GFP intensity within the targeted subnuclear spot (+/- SEM) (N cells: 1.125 J/m 2 = 16; 2.25 J/m 2 = 14; 4.5 J/m 2 = 19, 9 J/m 2 = 18; 18 J/m 2 = 18; 27 J/m 2 = 18). j) The average maximum cumulative NbSL3.3Q-GFP intensity after 10 minutes at various laser intensities (assay in Fig. ). k) Average mean GFP intensity +/− SEM of subnuclear spots of cells from retention experiment (Fig. ). Untreated siCtrl vs siATXN3, P97i siCtrl vs siATXN3. p-values (determined with Welch’s unpaired t-test) are indicated as ns (non-significant, p ≥ 0.05) * (p < 0.05) and **** (p < 0.0001). The lines inside the box of the box-and-whisker plots (Tukey style) in panel j and k indicate the median and the box itself encapsulates the interquartile range (IQR), with its lower and upper boundaries representing the first and third quartiles respectively. The whiskers extend from the box to cover the range within 1.5 times the IQR from the lower and upper quartiles, indicating the dispersion of the data. The lower whisker marks the minimum value, while the upper whisker denotes the maximum value, both excluding any outliers.

Article Snippet: Antibodies were sourced from the indicated manufacturers and used at 1:2,000 dilution unless otherwise stated: anti-GFP (Abcam, ab290), anti-GFP (Proteintech, 50430-2-AP; 1:5,000), anti-VCP/p97 (Proteintech, 10736-1-AP; 1:4,000), anti-ATXN3 (Proteintech, 13505-1-AP), anti-Ub (Biolegend, P4D1), anti-Ub K48-specific (Sigma, Apu2), anti-UBE2N (Invitrogen, 37-1100), anti-α-tubulin (CST, 3837; 1:5,000) and anti-GAPDH (Proteintech, 10494-1-AP; 1:5,000).

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Inhibition, Irradiation, Control, Stable Transfection, Whisker Assay, Dispersion