Fig. 5
From: The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity
The half-life of the p.I368N mutant PINK1 protein is reduced. a-b HeLa cells were simultaneously transfected with PINK1 siRNA and DNA constructs encoding for PINK1-V5 WT or p.I368N mutant and incubated with 100 μg/ml cycloheximide (CHX) alone (a) or pretreated with 10 μM CCCP for 2 h before incubation with cycloheximide (b). Cell lysates were analyzed by WB and V5/vinculin ratios were normalized to PINK1-V5 WT. Shown is the mean ± SEM from three independent experiments. Half-life (t1/2) of PINK1-V5 proteins was determined by curve fitting (one phase exponential decay). c Co-immunoprecipitation (IP) of the HSP90/CDC37 chaperone complex and the import channel of the mitochondrial outer membrane TOM40 with PINK1-V5. HeLa cells were transfected with PINK1-V5 WT, p.I368N or another PD-mutant PINK1 p.L347P as a control and left untreated or treated with 10 μM CCCP for 2 h. PINK1-V5 protein complexes were immunoprecipitated from cell lysates using V5 antibody conjugated to agarose. Representative WB is shown for input and IP samples probed with antibodies against V5 (PINK1), HSP90, CDC37, and TOM40. GAPDH served as a loading control. d Quantification of the co-immunoprecipitated proteins as performed in (c) is shown as the protein/V5 ratio. Data represent mean ± SEM from six independent experiments. Statistical significance was assessed by two-way ANOVA with Tukey’s post hoc; ***, p < 0.0005; **, p < 0.005; *, p < 0.05