Research Paper Volume 13, Issue 1 pp 77—88

Figure 2. Expression of Fbxo7 and its PD familial mutants resulted in an accumulation of endogenous Pink1 in a Pink1-Flag KI cell line. (A) a schematic illustration of an engineering strategy for generation of a Pink1-Flag KI cell line. Pink1 Hom1: Pink1 homology arm 1; NeoR: neomycin resistant gene; Pink1 Hom2: Pink1 homology arm 2. (B) confirmation of expression of Pink1-Flag in the positive Pink1-Flag KI clones with an anti-Flag antibody. The cells were treated by CCCP and MG-132. (C) expression of Fbxo7 and its PD familial forms led to accumulation of PF-Pink1. The plasmids harboring Fbxo7 or the PD associated mutations in Fbxo7 were transfected into the Pink1-Flag KI cells. The total cell lysates were analyzed by Western blots with anti-Flag and anti-Myc antibodies. After the relative level of Pink1 protein was obtained by normalizing of Pink to β-Actin, the relative ratio of Pink1 was obtained by normalization of the relative level of Pink1 from Fbxo7 transfected samples 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 = 0.0007. (D) KD of Fbxo7 caused a decrease of PF-Pink1. The Fbxo7 siRNA and control siRNA were transfected into the Pink1-Flag KI cells. 14 hours after transfection, the cells were treated by 10 μM MG-132 for 2 hours. The total cell lysates were analyzed by Western blots with anti-Flag and anti-Fbxo7 antibodies. After the relative level of Pink1 protein was obtained by normalizing to β-Actin, the relative ratio of Pink1 was obtained by normalization of the relative level of Pink1 from siFbxo7 transfected samples to the control transfected with siCtrl. The relative ratios were shown as mean ± SD; n = 3 independent experiments; unpaired 2-tailed Student’s t test; **p = 0.0058.