Fig. 5

OTUD4 deubiquitinates and stabilizes GSDME. A Representative images of silver-stained gels of immunoprecipitation proteins using anti-IgG and anti-Flag affinity beads. B Upper panel: list of candidate proteins from tandem affinity purification-coupled mass spectrometry. Lower panel: MS profiles of OTUD4. C Upper panel: schematic of protein extraction from parental or radioresistant SUNE1 and HK1 cells. Lower panel: Western blotting showed OTUD4 protein expression in parental or radioresistant SUNE1 and HK1 cells. D Interaction of GSDME with OTUD4. Co-IP was performed using cell lysates obtained from HEK293T cells co-transfected with Flag-GSDME and Myc-OTUD4. E Upper panel: schematic of OTUD4 truncations. Lower panel: schematic of GSDME truncations. F HEK293T cells were transfected with Flag-GSDME and indicated Myc-OTUD4 truncations. Lysates were then IP with Myc-beads for Western blotting. G HEK293T cells were transfected with Myc-OTUD4 and indicated Flag-GSDME truncations. Lysates were then IP with Flag-beads for Western blotting. H Indicated cells were transfected with OTUD4-expressing plasmid, or infected with lentiviral mediated shRNA against OTUD4 (#1 and #2). Whole-cell extracts were collected for Western blotting analysis. I Cells were harvested for Western blotting analysis at indicated time points after cycloheximide (CHX) treatment. J Quantification of GSDME protein levels normalized to GAPDH. K SUNE1 and HK1 cells were transfected with HA-Ub and Flag-GSDME with or without Myc-OTUD4. After treatment with MG132 (10 µM, 6 h) GSDME ubiquitination was measured. Results are presented as mean ± SD from three independent experiments