Figure 4.

Parkin-mediated regulation of Bax is cell-type independent. (a) Endogenous Bax expression in whole-cell lysates of HEK cells in a polyclonal line with stable expression of parkin (HEK-Parkin). HEK and HEK-Parkin cells either untreated (b) or treated with proteasome inhibitor (MG-132, 10 μM) for 6 h (c). Following lysis, whole-cell lysates and Bax IPs were probed by Western blot for ubiquitin. (d) Quantification of IPed ubiquitin levels from (b) and (c) plotted as a percentage of control (HEK). (e) Endogenous Bax expression in lysates from two monoclonal lines (M1 and M2) expressing differential levels of parkin. (f) HEK, HEK-Parkin_M1 and HEK-Parkin_M2 cells were treated with proteasome inhibitor (MG-132, 10 μM) for 6 h. Cells were lysed, and whole-cell lysates and Bax IPs were probed by Western blot for ubiquitin. (g) Quantification of IPed ubiquitin levels from b and c plotted as a percentage of control (HEK). (h) Quantitative RT-PCR was performed in HEK and HEK-Parkin_M2 cells to assess Bax mRNA levels. Graph is plotted as Bax mRNA levels normalized to control GAPDH mRNA levels. All bars represent mean±S.D., n=3. Asterisks indicate significant differences compared with control (P<0.05)