Fig. 4

VHL is an E3 ubiquitin ligase for Cx43, and MLKL competes with VHL for binding to Cx43. (A) Co-immunoprecipitation and Western blot to detect Cx43 ubiquitination in SH-SY5Y cells post transfection with Cx43(WT), Cx43(K303G), Ub, and VHL. The ubiquitination level of Cx43 was significantly decreased in VHL + Ub + Cx43 (K303G) transfection group (lane 5) in contrast to VHL + Ub + Cx43 (WT) transfection group (lane 4). (B) Co-immunoprecipitation and Western blot to detect the interaction among Cx43, MLKL, and VHL in SH-SY5Y cells. (C) Representative immunoblots of MLKL, pMLKL, Cx43, and VHL expression in SH-SY5Y cells post TSZ treatment, Cx43, MLKL(WT), and MLKL(S454A) transfection. D, E, F, G) Histogram showing the quantitative analyses of MLKL, pMLKL, Cx43, and VHL protein levels in SH-SY5Y cells after TSZ treatment and plasmids transfection (n ≥ 3 in each group). H) Co-immunoprecipitation and Western blot to detect the interaction among MLKL, pMLKL, VHL, and Cx43 in SH-SY5Y cells after TSZ treatment and plasmids transfection. Each bar represents mean ± standard deviation. ^*P < 0.05 vs. control group and ^#P < 0.05 vs. all groups except the control group. MLKL, mixed lineage kinase domain-like protein; Cx43, connexin 43; WT, wild type; MT, mutant; Ub, ubiquitin; VHL, von Hippel-Lindau; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TSZ, TNFα + Smac mimetic + ZVAD-FMK