Research Paper Volume 13, Issue 1 pp 77—88

Fbxo7 and Pink1 play a reciprocal role in regulating their protein levels

Expression of Fbxo7 and its PD familial mutants led to an accumulation of Pink1. The Pink1-Flag plasmid alongside Fbxo7-Myc or its mutated plasmids were co-transfected to three different cell lines (A: HEK293A, B: HeLa and C: SH-Sy5y). 24 hours after transfection, the cells were treated by 10 μM CCCP for 2 hours. The total cell lysates were subjected to Western blot analyses by anti-Flag antibody for Pink1-Flag, anti-Myc antibody for Fbxo7-Myc, and anti-β-Actin for β-Actin. After the relative level of FL-Pink1 and PF-Pink1 was obtained by normalizing of two forms of Pink1 to β-Actin respectively, the relative ratio of the two forms of Pink1 was obtained by normalization of the relative level of the two forms of Pink1 to the control only transfected with Pink1. The relative ratios were shown as mean ± SD; n = 3 independent experiments; individual 2-way ANOVAs with Tukey’s multiple comparisons test; ***p

Figure 1. Expression of Fbxo7 and its PD familial mutants led to an accumulation of Pink1. The Pink1-Flag plasmid alongside Fbxo7-Myc or its mutated plasmids were co-transfected to three different cell lines (A: HEK293A, B: HeLa and C: SH-Sy5y). 24 hours after transfection, the cells were treated by 10 μM CCCP for 2 hours. The total cell lysates were subjected to Western blot analyses by anti-Flag antibody for Pink1-Flag, anti-Myc antibody for Fbxo7-Myc, and anti-β-Actin for β-Actin. After the relative level of FL-Pink1 and PF-Pink1 was obtained by normalizing of two forms of Pink1 to β-Actin respectively, the relative ratio of the two forms of Pink1 was obtained by normalization of the relative level of the two forms of Pink1 to the control only transfected with Pink1. The relative ratios were shown as mean ± SD; n = 3 independent experiments; individual 2-way ANOVAs with Tukey’s multiple comparisons test; ***p<0.001.