Fig 1: K48-linked polyubiquitination mediated by SYVN1 promotes the interaction between SERPINA1E342K/ATZ and SQSTM1. (A) SQSTM1 interacts with SERPINA1E342K/ATZ through polyubiquitin chains that were conjugated by SYVN1. HEK 293T cells were transfected as indicated and treated with Baf A1 (20 nM) for 6 h. The interaction between SERPINA1E342K/ATZ and SQSTM1 was detected by coIP with anti-SERPINA1/AAT antibody followed by IB with anti-SQSTM1 antibody. (B) Schematic diagram of different ubiquitin mutants. (C) SYVN1-modified SERPINA1E342K/ATZ with Lys48-linked polyubiquitin chains is required for the interaction between SERPINA1E342K/ATZ and SQSTM1. HEK 293T cells were transfected with the plasmids that express SERPINA1E342K/ATZ, SYVN1-MYC, and HA-ubiquitin (WT; K48, K63, K48R or K63R mutants) and treated with Baf A1 (20 nM) for 6 h. The interaction between ubiquitinated SERPINA1E342K/ATZ and SQSTM1 was detected by coIP using anti-SERPINA1/AAT antibody followed by IB with anti-HA, anti-SERPINA1/AAT or anti-SQSTM1 antibodies. WT, wild type. (D) SYVN1 enhances the colocalization of K48-linked polyubiquitin chains and SERPINA1E342K/ATZ. HEK 293T cells were transfected and treated as indicated. Immunofluorescent staining was performed using antibodies against K48-ubiquitin (green), MYC (red), and SERPINA1E342K/ATZ (blue). The images were collected by using confocal microscopy. The extensive colocalization of SERPINA1E342K/ATZ and K48-linked polyubiquitin chains were indicated by arrows. Scale bar: 20 μm.
Fig 2: Overexpression of Hrd1 abolishes the protective effect of ATRA against UV-induced ROS production and cytotoxicity. Human fibroblasts were infected with ad-Hrd1 for 24 h, and then, exposed to UVA and UVB, and treated with ATRA for 24 h. (A) Fluorescence analyses were performed using a fluorescence microscope and quantified ROS production was detected. (B) Cell viability was assessed by WST-1 assay. Compared to control, **P<0.01; compared to UV, ##P<0.01; compared to UV+ATRA+Ad-GFP, &P<0.01. Hrd1, 3-hydroxy-3-methylglutaryl reductase degradation; ATRA, all-trans retinoic acid; UV, ultraviolet; ROS, reactive oxygen species.
Fig 3: HRD1 Silence could reserve OGD/R-induced injury to HBMECs at miR-602 deficiency situation. HBMECs were transfected with mimic of miR-602 inhibitor and HRD1 for 48 hours and then subjected to OGD/R treatment. (a) NRF2 and MCM3 relative expression level were detected by Western Blot. (b) NRF2 and HRD1 relative expression level at miR-602 deficiency situation was detected by Western Blot. (c) Luciferase activity of HRD1 3'-UTR wt and HRD1 3'-UTR mut with miR-602. (d) The effect of HRD1 silence on ROS level of HBMECs with low miR-602 level was detected by ROS detection assay. (e) The effect of HRD1 silence on relative NRF2/ARE transcription activity in HBMECs with low miR-602 level was detected by luciferase reporter assay. (f) The effect of HRD1 silence on the viability of HBMECs with low miR-602 level was detected by CCK-8. **P < 0.01.
Fig 4: Diagram of SYVN1 regulating GSDMD-mediated pyroptosis.Working model for SYVN1 promoting the activation of canonical and noncanonical inflammasomes-mediated pyroptosis. SYVN1 binds to GSDMD, promotes the K27-linked polyubiquitination of K203 and K204 lysine residues on GSDMD, resulting in the increasement of pyroptosis.
Fig 5: SYVN1 interacts with endogenous and exogenous GSDMD.A HEK293T cells co-transfected with pcDNA3.1-SYVN1-Myc and p3 × Flag-GSDMD were lysed and immunoprecipitated with anti-Flag antibody. Both the immunoprecipitates (IP) and whole-cell lysates (Input) were subjected to gel electrophoresis and analyzed by immunoblotting with anti-Myc and anti-Flag antibodies, respectively. B, C Immunoprecipitation analysis of THP-1 cells using anti-SYVN1 or anti-GSDMD antibodies. Immunoblotting of both IP and total proteins in THP-1 cells were performed using anti-SYVN1 and anti-GSDMD antibodies, respectively. D HEK293T cells co-transfected with pcDNA3.1-SYVN1-Myc and p3 × Flag-GSDMD were lysed and immunoprecipitated with anti-Flag antibody. The potential GSDMD-binding proteins in HEK293T cells were evaluated using Co-IP and MS analysis. The MS/MS spectrum of 71-AAEMEHLLER-80 is shown. Observed b- and y-ion series are indicated. E Immunostaining analysis of HEK293T cells after transfection with pcDNA3.1-SYVN1-Myc and p3 × Flag-GSDMD using anti-Flag and anti-Myc antibodies. Subcellular localization of Flag-GSDMD (green), Myc-SYVN1 (red), and DAPI (blue, a nucleus marker) were observed using confocal microscopy. Scale: 1 bar represents 10 µm. All experiments shown are representative of at least three independent experiments.
Supplier Page from Abcam for Anti-SYVN1/HRD1 antibody [EP7459]