Figure 3. Expression of Fbxo7 and its PD familial mutants had no effect on Pink1 ubiquitination. The Pink1-Flag plasmid along with Fbxo7-Myc or its mutated plasmids were co-transfected into HEK 293A cells. 24 hours after transfection, the cells were treated by 10 μM MG132 for 2 hours. The total cell lysates were subjected to immune-precipitation (IP) with anti-Flag antibody after 5-minute boiling. The IPed samples and the total cell lysates were analyzed by western blot with anti-ubiquitin antibody for ubiquitinated Pink1, anti-Flag antibody for Pink1-Flag, anti-Myc antibody for Fbxo7 and Bag2. The total cell lysates were analyzed by Western blots with anti-Flag and anti-Myc antibodies. After the relative level of ubiquitinated Pink1 protein was obtained by normalizing of ubiquitinated Pink to total Pink1, the relative ratio of ubiquitinated Pink1 was obtained by normalization of the relative level of ubiquitinated Pink1 from Fbxo7 or Bag2 transfected samples to the control transfected with Pink1 only (top panel from IP). 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.