Fig. 3
From: The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity
Full-length PINK1 protein and kinase activity are decreased in p.I368N mutant fibroblasts. a qRT-PCR was used to measure mRNA levels of PINK1 and housekeeping gene RPL27 in untreated cells and cells treated with 1 μM valinomycin for 24 h. Values of the PINK1/RPL27 mRNA ratio were normalized to untreated control cells. Error bars indicate mean ± SEM from six independent experiments (one-way ANOVA with Tukey’s post hoc; ns, not significant). b WT control and two PINK1 p.I368N fibroblasts were treated with 10 μM CCCP for 0, 2, 4 or 6 days and total lysates were analyzed by WB with the indicated antibodies. GAPDH served as a loading control. Slower migrating PINK1, PARKIN, and Mitofusin1 (MFN1) protein species indicate activated and/or modified forms and were only observed in control cells. p-Ser65-Ub signal increased in control cells over time but was undetectable in PINK1 p.I368N mutant cells. c Densitometric analysis of full-length PINK1 protein from (b). Values were normalized to controls cells treated for 6 days with CCCP. Error bars indicate mean ± SEM from four independent experiments. Values were tested for statistical significance compared to control cells (two-way ANOVA with Tukey’s posthoc test, *, p < 0.05, ***, p < 0.0005). d Representative images of fibroblasts upon treatment with 1 μM valinomycin. Cells were stained with anti p-Ser65-Ub (green) and TOM20 (red). Nuclei are shown in blue (Hoechst). Scale bars indicate 10 μm. Upon treatment, p-Ser65-Ub is induced only in control, but not in PINK1 p.I368N fibroblasts. e p-Ser65-Ub signal upon valinomycin treatment was quantified by HCI. Control and PINK1 p.I368N fibroblasts were incubated with 1 μM valinomycin for 0, 4, 8 and 24 h. Cells were fixed and stained with p-Ser65-Ub antibodies and a nuclear counterstain (Hoechst). Mean intensity of p-Ser65-Ub in a cytoplasmic ring around the nucleus was measured and normalized to values of untreated and 24 h valinomycin treated control cells. Error bars indicate mean ± SEM from three independent experiments (two-way ANOVA with Tukey’s post hoc; ***, p < 0.0005)