Figure 4

The Parkin-Hsc70 complex regulates the stability of the p62 protein under hypoxia. (A) Parkin in 3 × FLAG-pcDNA4 was transfected into Hep3B cells, cells were then incubated under normoxia or hypoxia (O₂ = 1%) for 12 h, and p62 protein levels were examined by immunoblotting. (B) FLAG-Parkin was overexpressed in Hep3B cells, cultured under normoxia or hypoxia for 12 h, and immunoprecipitated with the anti-FLAG antibody. The band indicated by the arrow was analyzed by LC–MS. (C) Hep3B cells were transfected with the FLAG-p62, cell lysates were immunoprecipitated with an anti-FLAG antibody, and immunoprecipitated proteins were subjected to an immunoblotting analysis with anti-FLAG, anti-Hsc70, and anti-Parkin antibodies. (D) Hep3B cells were transfected with FLAG-Hsc70, incubated under normoxia or hypoxia for 12 h, and p62 protein levels were examined by immunoblotting. (E) FLAG-Hsc70 was transfected into Hep3B cells, incubated under normoxia or hypoxia for 12 h, treated with 5 µM MG132, and p62 protein levels were examined by immunoblotting. (F) FLAG-p62 was transfected with siRNA-Control, siRNA-Parkin, or siRNA-Hsc70, grown under normoxia or hypoxia for 12 h in the presence of 5 µM MG132, and cell lysates were immunoprecipitated using an anti-FLAG antibody, and subjected to immunoblotting. (G) Hep3B cells were transfected with pcDNA-Parkin-Venus and the cellular co-localization of p62 (magenta), Parkin (green), Hsc70 (red), and DAPI (blue) was examined by immunofluorescence, scale bar: 10 μm. The histogram shows the fluorescence intensity of channels along the white box in the merged image. (H) Hep3B cells were transfected with siRNA-Control, siRNA-Parkin (Parkin K.D.), or siRNA-Hsc70 (Hsc70 K.D.), incubated under normoxia or hypoxia for 12 h, and p62 protein levels were examined by immunoblotting. (I) pcDNA-Parkin was co-transfected with siRNA-Control or siRNA-Hsc70, incubated under hypoxia for 12 h, and subjected to immunoblotting to assess p62 protein levels. (J) pcDNA-Hsc70 was co-transfected with siRNA-Control or siRNA-Parkin, incubated under hypoxia for 12 h, and subjected to immunoblotting. Data are presented as the mean ± S.D. of triplicate experiments. N.S. not significant, *p < 0.05, ***p < 0.001 versus the indicated group, calculated with a one-way ANOVA with Tukey’s post-hoc test.