Figure 6.

The PARL-PGAM5 axis is involved in PHB2-mediated PINK1 stabilization. (a) HeLa cells were transfected with negative control (NC) or siRNA against PGAM5 for 72 h, and then treated with OA for 2 h. Cell lysates were analyzed by western blotting with the antibodies against PINK1, PGAM5, or TUBB. (b) HeLa cells stably expressing PINK1-GFP were transiently transfected with control (empty vector) or PGAM5-MYC. Cells were treated 48 h later with or without OA for 2 h. Then cell lysates were analyzed by western blotting with anti-GFP, anti-MYC, or anti-TUBB antibodies. (c) 293T cells were transiently co-transfected with PGAM5-MYC and PINK1-GFP. Cells were then harvested 48 h later for the isolation of mitochondria. Mitochondria were treated with the indicated doses of proteinase K for 30 min on the ice and then were analyzed by western blotting with anti-GFP, anti-MYC, anti-TOMM20, or anti-TIMM23 antibodies. (d) 293T cells were transiently co-transfected with control (empty vector) and PINK1-GFP, or PGAM5-MYC and PINK1-GFP. Cells were treated 48 h later with CCCP (10 μM) for 4 h. Cell lysates were used for immunoprecipitation with Dynabeads Protein G pre-coupled with anti-MYC antibody at 4°C overnight, followed by western blotting with anti-GFP or anti-MYC antibodies. (e) 293T cells were transiently transfected with control (empty vector) or PGAM5-MYC for 48 h, and then treated with CCCP for 4 h. Cells lysates were used for immunoprecipitation with anti-MYC antibody coupled Dynabeads Protein G at 4°C overnight, followed by western blotting using anti-PHB, anti-PHB2, or anti-MYC antibodies. (f) 293 cells were treated with or without CCCP for 4 h. Cells lysates were used for immunoprecipitation with Dynabeads Protein G coupled with anti-PHB2 antibody at 4°C overnight, followed by western blotting with the indicated antibodies. (g) 293T cells were transiently transfected with empty vector or plasmids coding for PHB2-FLAG and PGAM5-MYC for 48 h, and then treated with CCCP (10 μM) for the indicated time. Cells lysates were used for immunoprecipitation with anti-FLAG M2 affinity gel at 4°C overnight. Immunoprecipitates and cells lysates were analyzed by western blotting with anti-FLAG, anti-MYC, or anti-PARL antibodies. (h-i) HeLa cells expressing PINK1-GFP were transfected with control (empty vector), PGAM5-MYC, or MTS (AIFM1)-S-PGAM5-MYC for 48 h, and then treated with OA for 2 h. Cell lysates were analyzed by western blotting with anti-GFP, anti-MYC, or anti-TUBB antibodies. (i) Quantification of the ratio between full-length PINK1-GFP and cleaved-PINK1-GFP. Error bars indicate the mean ±SD of 3 independent experiments, **p < 0.01.