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. 2022 Dec 18;10(5):2205483. doi: 10.1002/advs.202205483

Figure 6.

Figure 6

PINK1 phosphorylates Rab22a‐NeoF1 at Ser120 to promote its turnover by STUB1. a) 293T cells co‐transfected with the indicated plasmids for 48 h were analyzed by western blot. The experiments were repeated three times independently with similar results. b) The phosphorylation mass spectrometry of Rab22a‐NeoF1 protein as described in methods. c) 293T cells co‐transfected with the indicated plasmids for 48 h were lysed and immunoprecipitated using anti‐FLAG beads followed by western blot analysis. The experiments were repeated three times independently with similar results. d) Rab22a‐NeoF1‐SFB or its S120A mutant protein were purified from 293T cells and incubated with or without the purified V5 tagged PINK1 kinase in vitro as described in the methods and then analyzed by western blot. The experiments were repeated three times independently with similar results. e,f) Immunoprecipitation was performed using ZOS overexpression or knockout PINK1 cells with IgG and hAb RAD5‐8 then were analyzed by western blot. The experiments were repeated three times independently with similar results. g) The indicated stable cells were analyzed by western blot. The experiments were repeated three times independently with similar results. h,i) Quantification analyses of migration and invasion assays using the indicated stable cell lines in (g). The depicted results are the averages of at least three independent experiments. The data are presented as the mean ± SD. A two‐sided unpaired student's t‐test was performed, and p values are shown. j–l) 293T cells co‐transfected with the indicated plasmids for 48 h were lysed and immunoprecipitated using anti‐FLAG beads followed by western blot analysis. The experiments were repeated three times independently with similar results. m) 293T cells stably expressing Rab2a‐NeoF1 wild type or its K112A mutant as indicated were transfected with STUB1‐Myc for 48 h, treated with cycloheximide (CHX: 20 µg mL−1) for the indicated time, and then subjected to western blot. n) Quantitation of the Rab22a‐NeoF1 protein levels in (m). The experiments were repeated three times independently with similar results.