Fig. 4
PTEN-L impairs Parkin E3 ligase activity and reduces pSer65-Parkin. a YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for indicated hours. YFP-Parkin was immunoprecipitated with anti-GFP antibody. Immunoprecipitants (IPs) and whole-cell lysates (WCLs) were analyzed for YFP-Parkin, mitofusin-2 (MFN2), Tom20, PTEN-L, PTEN and tubulin. b YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with O/A (10 nM and 100 nM) for indicated hours and the WCLs were analyzed by immunoblotting as indicated. c Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with CCCP (5 µM) for indicated hours and the WCLs were analyzed by immunoblotting as indicated. d Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with O/A (10 nM and 100 nM) for indicated hours and the WCLs were analyzed by immunoblotting as indicated. e The MS/MS spectra of the Parkin peptide containing phospho-Ser65. YFP-Parkin-HeLa cells were treated with CCCP (10 µM) for 4 h. YFP-Parkin was pulled down with GFP beads and subjected to MS/MS analysis. f pSer65-Parkin was quantified using MS-based relative quantification analysis in YFP-Parkin-HeLa cells with or without PTEN-L stable expression after CCCP (10 µM) and O/A (25 nM and 250 nM) treatment. Data are presented as mean ± SD from 3 independent experiments. **P < 0.01 (Student’s t-test)
