Fig. 3

Interaction between MLKL and Cx43 inhibits the K48-linked ubiquitination of Cx43 in necroptotic SH-SY5Y cells. (A) Co-immunoprecipitation to detect the interaction between Cx43, MLKL(WT), and MLKL(S454A) in SH-SY5Y cells. (B) Co-immunoprecipitation and Western blot to detect Cx43 ubiquitination in SH-SY5Y cells post MLKL(WT) and MLKL(S454A) transfection. The ubiquitination level of Cx43 was significantly upregulated in Cx43 + Ub + MLKL(S454A) transfection group (lane 4) in contrast to Cx43 + Ub + MLKL(WT) transfection group (lane 3). (C) Co-immunoprecipitation and Western blot to detect K48-linked ubiquitination of Cx43 in SH-SY5Y cells post MLKL(WT) and MLKL(S454A) transfection. The K48-linked ubiquitination level of Cx43 was significantly upregulated in Cx43 + MLKL(S454A) transfection group (lane 5) in contrast to Cx43 + MLKL(WT) transfection group (lane 4). (D) Viability of SH-SY5Y cells after transfection with MLKL(WT), MLKL(S454A), and Cx43(WT) plasmids (n ≥ 3 in each group). (E) Release of LDH in SH-SY5Y cells after transfection with MLKL(WT), MLKL(S454A), and Cx43(WT) plasmids (n ≥ 3 in each group). (F) After transfecting SH-SY5Y cells with MLKL(WT), MLKL(S454A), and Cx43(WT) plasmids, representative photographs of flow cytometry analysis of necroptotic cells. (G) After transfecting SH-SY5Y cells with MLKL(WT), MLKL(S454A). and Cx43(WT) plasmids, quantitative analysis of necroptotic cells by flow cytometry analysis (n ≥ 3 in each group). 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; TSZ, TNFα + Smac mimetic + ZVAD-FMK; PI, propidium iodide