Fig. 4

PARL-cleaved forms of PINK1 WT and p.I368N are similarly stabilized upon proteasome inhibition. a Control and PINK1 p.I368N fibroblasts were pretreated with 500 nM epoxomicin for 1 h followed by 10 μM CCCP treatment for additional 24 h, as indicated. Only PARL-cleaved PINK1 p.I368N (~52 kDa, black arrowhead), but not the full-length form (~63 kDa, white arrowhead) accumulated with epoxomicin ± CCCP in mutant fibroblasts. Anti-p-Ser65-Ub and total Ub antibodies were used as controls for CCCP and epoxomicin treatment, respectively. GAPDH served as a loading control. b WB quantification of full-length (top) and PARL-cleaved (bottom) PINK1 species from control and p.I368N mutant fibroblasts as performed in (a). Values of the full-length PINK1/GAPDH ratio were normalized to control cells treated only with CCCP. Values of the cleaved PINK1/GAPDH ratios were normalized to control cells treated only with epoxomicin. Error bars indicate mean ± SEM from three independent experiments (two-way ANOVA with Tukey’s post hoc; *, p < 0.05; ***, p < 0.0005). c Fibroblasts were pretreated with 500 nM epoxomicin for 1 h followed by 1 μM valinomycin treatment for additional 24 h, as indicated. Cells were homogenized in fractionation buffer and proteins were sequentially extracted into soluble (RIPA) and insoluble (urea) fractions and were analyzed by WB using PINK1 antibody. Vinculin served as a loading control. White and black arrowheads indicate PINK1 full-length (mostly in soluble fraction) and the PARL-cleaved form (mostly in insoluble fraction), respectively. Black arrow indicates an intermediate (probably MPP-cleaved) form of PINK1 (~60 kDa), which could be detected in the “insoluble fraction” after epoxomicin treatment. d HeLa cells were transfected with control or PINK1 siRNA together with constructs encoding for PINK1-V5 (WT, p.I368N, p.F104A, or the double mutant p.F104A + p.I368N). Cells were left untreated or treated with 10 μM CCCP for 2 h. Levels of full-length and cleaved PINK1 were analyzed by WB using anti-V5 and anti-PINK1 antibodies. Vinculin served as a loading control. e Densitometric analysis of the full-length (top) and PARL-cleaved (bottom) PINK1 species from (d). Values of the full-length PINK1-V5/vinculin ratio were normalized to CCCP-treated cells expressing WT PINK1-V5. Values of the cleaved PINK1-V5/vinculin ratio were normalized to untreated cells expressing PINK1-V5 p.F104A mutant. Error bars indicate mean ± SEM from three independent experiments (two-way ANOVA with Tukey’s post hoc; ***, p < 0.0005)