ha tagged ub wt ha ub  (Addgene inc)

Journal: bioRxiv

Article Title: IFI207 promotes antiviral responses by modulating STING ubiquitination and degradation

doi: 10.64898/2026.03.05.709838

Figure Lengend Snippet: IFI207 reduces K63-linked ubiquitination on STING following DMXAA stimulation. A) BMDMs from the indicated mice were treated with 100 µg/mL DMXAA for 1 hr. An anti-K63-linkage-specific polyubiquitin antibody was used to immunoprecipitate cell extracts. The immunoprecipitates were then analyzed by western blotting using anti-STING and anti-tubulin antibodies. Shown to the right is quantification of 3 independent experiments ± SD. Two-way ANOVA was used to determine significance. **, P ≤0.03; ns, not significant. B) HEK293T cells were transfected with either HA-tagged Ub-WT or -K63 expression plasmids along with Flag-tagged STING and V5-tagged IFI207. 24 hr after transfection, the cells were treated with 100 μg/ml DMXAA for 2 hr. Immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. To quantify, the ubiquitin signal was normalized to the STING signal and then the EV (4 hr) sample was set to 1. Shown below is quantification of the average of 3 independent experiments ± SD. Two-way ANOVA was used to determine significance. *, P ≤0.01; ns, not significant. C) HEK293T cells were transfected with HA-tagged Ub-63 plasmids along with Flag-tagged STING and V5-tagged IFI207. 24 hr after transfection, the cells were pre-treated with TAK243 for 30 mins and then stimulated with DMXAA for 4 hr. Immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. Shown to the right is quantification of the average of 3 independent experiments ± SD. Two-way ANOVA was used to determine significance. **, P ≤0.0008; ns, not significant.

Article Snippet: The HA-tagged Ub-WT HA-Ub (#17608) and HA-Ub (K63 only) (#17606) plasmids were from Addgene.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Expressing, Immunoprecipitation

Journal: Autophagy

Article Title: Regulation of pexophagy by a novel TBK1-MARCHF7-PXMP4-NBR1 axis in PEX1-depleted HeLa cells

doi: 10.1080/15548627.2025.2593585

Figure Lengend Snippet: E3 ubiquitin ligase MARCHF7 mediates PEX1 depletion-induced pexophagy. ( A and B ) wild-type (WT) and ATG5 -knockout (KO) HeLa cells were transiently transfected with either scrambled siRNA (SC) or validated siRNA targeting PEX1 (si PEX1 ) for 72 h. Cells were then immunostained with an anti-ABCD3 antibody. The number of peroxisomes per cell was quantified ( n ≥200). ( C and D ) HeLa cells were transiently transfected with Sc or si PEX1 for 24 h, followed by treatment with bafilomycin A 1 (Baf.A1, 10 nM) for an additional 48 h. Cells were then stained with an anti-ABCD3 antibody. The number of peroxisomes per cell was quantified ( n ≥200). ( E and F ) HeLa cells were transiently transfected with Sc, si PEX1 , or siRNA targeting MARCHF7 (si MARCHF7 ) for 72 h. Cells were then fixed, immunostained with an anti-ABCD3 antibody, and imaged using confocal microscopy. The number of peroxisomes per cell was quantified ( n ≥200). ( G and H ) HeLa cells were co-transfected with mCherry-SEpHluorin-SKL and either si PEX1 or si MARCHF7 for 72 h. Cells were imaged using fluorescence microscopy, and the proportion of mCherry + SEpHluorin + peroxisomes and mCherry + SEpHluorin − pexolysosomes was calculated ( n ≥100). ( I and J ) HeLa cells were transfected with Sc or si PEX1 for 72 h. Cells were then fixed, immunostained with anti-ABCD3 and anti-MARCHF7 antibodies, and imaged using confocal microscopy. Pearson’s correlation coefficient was used to quantify the colocalization between ABCD3 and MARCHF7. The graph represents the colocalization analysis results obtained using the Coloc2 plugin in Fiji, based on Pearson’s correlation coefficient ( n ≥100). Scale bar: 20 µm. Data are presented as mean ± standard error of the mean (SEM); **p < 0.001.

Article Snippet: The HA-tagged ubiquitin WT (HA-UB) plasmid was obtained from Addgene (17608; deposited by Ted Dawson).

Techniques: Ubiquitin Proteomics, Knock-Out, Transfection, Staining, Confocal Microscopy, Fluorescence, Microscopy

Journal: Autophagy

Article Title: Regulation of pexophagy by a novel TBK1-MARCHF7-PXMP4-NBR1 axis in PEX1-depleted HeLa cells

doi: 10.1080/15548627.2025.2593585

Figure Lengend Snippet: MARCHF7 mediates PXMP4 (lysine 20) ubiquitination dependent pexophagy in PEX1-depleted cells. ( A and B ) HeLa cells were transiently transfected with Sc or si PEX1 in combination with siRnas targeting peroxisomal membrane proteins for 72 h. Cells were then immunostained with an anti-ABCD3 antibody, and the number of peroxisomes per cell was quantified ( n ≥100). Scale bar: 20 µm. ( C and D ) HeLa cells were co-transfected with mCherry-SEpHluorin-SKL and either si PEX1 or si PXMP4 for 72 h. Cells were imaged using fluorescence microscopy, and the proportion of mCherry + SEpHluorin + puncta and mCherry + SEpHluorin − puncta was calculated ( n ≥100). ( E ) HeLa cells were transfected with Sc or si PEX1 for 72 h, harvested, and subjected to immunoprecipitation using anti-MARCHF7 antibodies conjugated to agarose beads. Samples were analyzed by western blotting with the indicated antibodies. ( F ) HeLa cells were transfected with si PEX1 or si MARCHF7 along with HA and HA-ubiquitin (HA-UB) for 48 h, followed by treatment with MG132 (10 µM) for 12 h. Cells were harvested and subjected to immunoprecipitation using anti-PXMP4 antibodies conjugated to agarose beads, and samples were analyzed by western blotting with the indicated antibodies. ( G ) HeLa cells were transfected with Sc, si PEX1 , Flag-tagged PXMP4 K/R mutants (PXMP4 wt, PXMP4 K20R , PXMP4 K31R , or PXMP4 K207R ) in combination with HA-UB for 48 h, followed by MG132 treatment (10 µM) for 12 h. Cells were then harvested and subjected to immunoprecipitation using anti-Flag antibodies conjugated to agarose beads, and samples were analyzed by western blotting with the indicated antibodies. ( H ) HeLa cells were transiently transfected with Sc or si PEX1 in combinations with Flag (empty vector, ev), PXMP4 wt or PXMP4 K20R mutant for 48 h. After, the cells were harvested and subjected to immunoprecipitation by using agarose-conjugated anti-Flag antibody. The samples were analyzed by western blotting with the indicated antibodies. Scale bar: 20 µm. Data are presented as mean ± SEM; **p < 0.001.

Article Snippet: The HA-tagged ubiquitin WT (HA-UB) plasmid was obtained from Addgene (17608; deposited by Ted Dawson).

Techniques: Ubiquitin Proteomics, Transfection, Membrane, Fluorescence, Microscopy, Immunoprecipitation, Western Blot, Plasmid Preparation, Mutagenesis

Journal: Autophagy

Article Title: Regulation of pexophagy by a novel TBK1-MARCHF7-PXMP4-NBR1 axis in PEX1-depleted HeLa cells

doi: 10.1080/15548627.2025.2593585

Figure Lengend Snippet: TBK1 phosphorylation is induced by ros accumulation following PEX1 depletion. ( A and B ) HeLa cells were transfected with Sc or si PEX1 for 72 h, treated with or without nac (1 mM) for 48 h, and incubated with DCFH-DA (10 µM, 15 min). Cells were imaged, and fluorescence intensity was quantified using ImageJ. Data represent mean ± SEM from 3 independent experiments; 100 cells were analyzed per condition in each experiment. Scale bar: 50 µm. ( C ) HeLa cells were transfected with Sc or si PEX1 for 72 h, treated with NAC (1 mM) for 48 h, and analyzed by western blotting with the indicated antibodies. ( D ) HeLa cells were transfected with Sc or si PEX1 for 72 h, treated with NAC (1 mM) for 48 h, and subjected to immunoprecipitation using anti-MARCHF7 antibodies conjugated to agarose beads. Samples were analyzed by western blotting with the indicated antibodies. ( E ) HeLa cells were transfected with si PEX1 along with HA and HA-UB for 48 h, then treated with MG132 (10 µM) for 12 h, with or without NAC (1 mM) for 24 h. Cells were subjected to immunoprecipitation using anti-PXMP4 antibodies conjugated to agarose beads, and samples were analyzed by western blotting. ( F and G ) HeLa cells were transfected with Sc or si PEX1 for 72 h, treated with or without NAC (1 mM) for 48 h, fixed, immunostained with an anti-ABCD3 antibody, and imaged using confocal microscopy. The number of peroxisomes per cell was quantified ( n ≥ 200). Scale bar: 20 µm. Data are presented as mean ± SEM; **p < 0.001.

Article Snippet: The HA-tagged ubiquitin WT (HA-UB) plasmid was obtained from Addgene (17608; deposited by Ted Dawson).

Techniques: Phospho-proteomics, Transfection, Incubation, Fluorescence, Western Blot, Immunoprecipitation, Confocal Microscopy

Journal: Autophagy

Article Title: Regulation of pexophagy by a novel TBK1-MARCHF7-PXMP4-NBR1 axis in PEX1-depleted HeLa cells

doi: 10.1080/15548627.2025.2593585

Figure Lengend Snippet: Influence of the TBK1-MARCHF7-PXMP4-NBR1 axis on pexophagy induced by PEX1 depletion. ( A and B ) HeLa cells were transiently transfected with scrambled siRNA (Sc), PEX1 siRNA (si PEX1 ), validated NBR1 siRNA (si NBR1 ), or validated SQSTM1 siRNA (si SQSTM1 ) for 72 h. Cells were fixed, immunostained with an anti-ABCD3 antibody, and imaged using confocal microscopy. The number of peroxisomes per cell was quantified ( n ≥100). ( C ) HeLa cells were transiently transfected with si PEX1 along with HA and HA-UB for 48 h, followed by treatment with MG132 (10 µM) for 12 h, with or without GSK8612 (10 µM) for 24 h. Cells were harvested and subjected to immunoprecipitation using anti-PXMP4 antibodies conjugated to agarose beads. Samples were analyzed by western blotting with the indicated antibodies. ( D and E ) HeLa cells were transiently transfected with Sc, si PEX1 , si MARCHF7 , or si PXMP4 for 72 h, then fixed and immunostained with anti-ABCD3 (peroxisome) and anti-NBR1 antibodies. Cells were imaged using confocal microscopy. Pearson’s correlation coefficient was used to quantify the colocalization between peroxisomes and NBR1. The graph represents the colocalization analysis results obtained using the coloc 2 plugin in Fiji, based on Pearson’s correlation coefficient ( n ≥100). Scale bar: 20 µm. Data are presented as mean ± standard error of the mean (SEM); * p < 0.01, ** p < 0.001.

Article Snippet: The HA-tagged ubiquitin WT (HA-UB) plasmid was obtained from Addgene (17608; deposited by Ted Dawson).

Techniques: Transfection, Confocal Microscopy, Immunoprecipitation, Western Blot

Journal: Journal of Virology

Article Title: Oxidative stress sensor Keap1 recognizes HBx protein to activate the Nrf2/ARE signaling pathway, thereby inhibiting hepatitis B virus replication

doi: 10.1128/jvi.01287-23

Figure Lengend Snippet: HBx induces polyubiquitylation of Nrf2 via K6-linked polyubiquitin chains. (A) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pCAG-Keap1, and pRK5-HA-Ub, together with increasing amounts of pEF1A-HBx-Myc-His6. At 48 h after transfection, the cells were harvested by treatment with 25 µM MG132 for 8 h. The cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-HA polyclonal antibody (PAb) (first panel) or anti-Nrf2 monoclonal antibody (MAb) (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-Keap1 MAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MAb (seventh panel). The level of GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pEF1A-HBx-Myc-His6, and pRK5-HA-Ub. At 48 h after transfection, the cells were harvested by treatment with 25 µM MG132 for 8 h. The cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (first panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-Keap1 MAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). The level of GAPDH served as a loading control. (C) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pCAG-Keap1, pEF1A-HBx-Myc-His6, and the indicated HA-tagged Ub plasmids. At 48 h after transfection, the cells were harvested by treatment with 25 µM MG132 for 8 h. The cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (first panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-Keap1 MAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). The level of GAPDH served as a loading control. (D) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h after siRNA-transfection, cells were co-transfected with pCAG-FLAG-Nrf2, pCAG-Keap1, and pEF1A-HBx-Myc-His6 together with pRK5-HA-Ub or pRK5-HA-Ub-K6. At 48 h after transfection, the cells were harvested by treatment with 25 µM MG132 for 8 h. The cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (first panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-Keap1 MAb (fifth panel), anti-HUWE1 PAb (sixth panel), anti-c-Myc MAb (seventh panel), or anti-GAPDH MAb (eighth panel). The level of GAPDH served as a loading control.

Article Snippet: The N-terminal HA-tagged Ub expression plasmids pRK5-HA-Ub-WT, pRK5-HA-Ub-K6, pRK5-HAUb-K11, pRK5-HA-Ub-K27, pRK5-HA-Ub-K29, pRK5-HA-Ub-K33, pRK5-HA-Ub-K48, and pRK5-HA-Ub-K63 (all from Addgene, Watertown, MA, USA) and the plasmids pGL4.10-HBpg-Ce (A1676C/C1678A) ( 41 ) and pUC19-HBV-C-AT_JPN(ΔHBx) ( 53 ) were also used. . Antibodies The mouse monoclonal antibodies (MAbs) used in this study were anti-Nrf2 MAb (A-10; sc-365949; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Keap1 MAb (G-2; sc-365626; Santa Cruz Biotechnology), anti-c-Myc MAb (9E10; sc-40; Santa Cruz Biotechnology), anti-HBc MAb (clone 7B2, culture supernatant of the hybridoma) ( 54 , 55 ), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MAb (014-25524; FUJIFILM Wako Pure Chemical Industries).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Theranostics

Article Title: H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts

doi: 10.7150/thno.51245

Figure Lengend Snippet: COL6A1 interacted with E3 ligase SOCS5 to promote STAT1 degradation. A. Co-localization of COL6A1 and STAT1 in ESCC cells were examined by using confocal microscopy (scale bar, 10 µm). B. The interaction of GFP-STAT1 and Flag-COL6A1 wide type, VWFA1, VWFA2, VWFA3 domains deletion mutations were detected by co-immunoprecipitation in 293T cells. C. COL6A1 was transfected into the OS cells in the presence of cycloheximide (CHX, 200 µg/mL) for indicated times. Cell lysates were immunoblotted by antibodies as indicated. The data were quantified using Image J software. D. STAT1 ubiquitination was detected by immunoprecipitation with anti-STAT1 antibody and immunoblotting with an anti-Ub antibody. E. The interaction of STAT1, COL6A1 and SOCS5 was detected by co-immunoprecipitation in U2OS. F. SOCS5 decreased STAT1 protein. U2OS cells were transfected with Flag-SOCS5 or Flag-SOCS5 R406K as well as control or COL6A1 transfection. The protein expression level of STAT1 was assayed by western blot. G . The cells expressing wide type SOCS5 were treated with CHX. The protein levels of STAT1 and SOCS5 were analyzed by western blot. H . Knockdown SOCS5 increased STAT1 protein. U2OS cells were transfected with si-SOCS5 as well as control or COL6A1 transfection. I . U2OS cells were transfected with control or SOCS5 siRNAs treated with CHX (200 µg/mL), the protein levels of STAT1 and SOCS5 were analyzed by western blot. J. SOCS5 ubiquitylates STAT1. U2OS cells were transfected with indicated plasmids or siRNA for 48 h. Cell lysates were immunoprecipitated with anti-GFP and analyzed by immunoblotting with indicated antibodies. K . U2OS cells were transfected with indicated plasmids. lysates were immunoprecipitated with anti-GFP, and western blots were performed to analyze the presence of indicated proteins and levels of ubiquitination. Data represent the mean ± SD of 3 separate determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t test.

Article Snippet: The expression vectors containing the p300, c-Jun, COL6A1, SOCS5, mutant SOCS5, HA-tagged Wild type (wt) Ubiqution (Ub), HA-tagged K11R Ub, HA-tagged K48R Ub and HA-tagged K63R Ub plasmids were purchased from Vigene (Shanghai, China).

Techniques: Confocal Microscopy, Immunoprecipitation, Transfection, Software, Ubiquitin Proteomics, Western Blot, Control, Expressing, Knockdown