Figure 5
G6PD is involved in PINK1 stabilization and ubiquitin phosphorylation. (a−f) Detection of p-Ub and PINK1 protein levels by immunoblotting analysis. (a) WT and G6PD KO HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for the indicated time points and subjected to immunoblotting analysis. (b) G6PD KO HeLa 3+ cells were transfected with WT G6PD for 24 h. Cells were then treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h and subjected to immunoblotting analysis. RE: reconstituted. (c) WT, G6PD KO, and PGLS KO HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h. Lysates were subjected to immunoblot analysis. (d) HeLa 3+ cells were pretreated with G6PD inhibitors DHEA (500 μmol/L) or 6-AN (1 mmol/L) for 1 h before O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 2 h. G6PD KO cells were used as a negative control. (e) HeLa 3+ cells were pretreated with BAY (10 μmol/L), PAR (25 μmol/L), or WED (50 μmol/L) for 1 h followed by O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 2 h. G6PD KO cells were used as a negative control. Loading control is shared with Fig. 4d. (f) WT and G6PD KO HeLa 3+ cells were pretreated with AG1 (20 μmol/L) for 1 h before O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 4 h. (g) Visualization of mCherry-Parkin translocation by live cell imaging. HeLa 3+ (WT and PINK1 KO) cells were pretreated with AG1 (20 μmol/L) for 1 h before O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 2 h. Images were taken using a Leica fluorescence microscope. Scale bar: 20 μm. (h) Detection of mitochondrial protein levels by immunoblotting. HeLa 3+ (WT and PINK1 KO) cells were pretreated with AG1 (20 μmol/L) for 1 h before O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 4 h. Lysates were subjected to immunoblotting analysis with the indicated antibodies. (i) Mitochondrial protein levels in G6PD-overexpressing cells. G6PD KO and PINK1 KO HeLa 3+ cells were transfected with WT G6PD for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h before lysis and immunoblotting analysis. (j) Cleaved PINK1 levels observed by immunoblotting analysis. HeLa 3+ (WT and G6PD KO) cells were pretreated with MG132 (10 μmol/L) for 1 h before being treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h. Lysates were immunoblotted with the indicated antibodies.

G6PD is involved in PINK1 stabilization and ubiquitin phosphorylation. (a−f) Detection of p-Ub and PINK1 protein levels by immunoblotting analysis. (a) WT and G6PD KO HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for the indicated time points and subjected to immunoblotting analysis. (b) G6PD KO HeLa 3+ cells were transfected with WT G6PD for 24 h. Cells were then treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h and subjected to immunoblotting analysis. RE: reconstituted. (c) WT, G6PD KO, and PGLS KO HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h. Lysates were subjected to immunoblot analysis. (d) HeLa 3+ cells were pretreated with G6PD inhibitors DHEA (500 μmol/L) or 6-AN (1 mmol/L) for 1 h before O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 2 h. G6PD KO cells were used as a negative control. (e) HeLa 3+ cells were pretreated with BAY (10 μmol/L), PAR (25 μmol/L), or WED (50 μmol/L) for 1 h followed by O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 2 h. G6PD KO cells were used as a negative control. Loading control is shared with Fig. 4d. (f) WT and G6PD KO HeLa 3+ cells were pretreated with AG1 (20 μmol/L) for 1 h before O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 4 h. (g) Visualization of mCherry-Parkin translocation by live cell imaging. HeLa 3+ (WT and PINK1 KO) cells were pretreated with AG1 (20 μmol/L) for 1 h before O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 2 h. Images were taken using a Leica fluorescence microscope. Scale bar: 20 μm. (h) Detection of mitochondrial protein levels by immunoblotting. HeLa 3+ (WT and PINK1 KO) cells were pretreated with AG1 (20 μmol/L) for 1 h before O/A (5 μmol/L and 1 μmol/L, respectively) treatment for 4 h. Lysates were subjected to immunoblotting analysis with the indicated antibodies. (i) Mitochondrial protein levels in G6PD-overexpressing cells. G6PD KO and PINK1 KO HeLa 3+ cells were transfected with WT G6PD for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h before lysis and immunoblotting analysis. (j) Cleaved PINK1 levels observed by immunoblotting analysis. HeLa 3+ (WT and G6PD KO) cells were pretreated with MG132 (10 μmol/L) for 1 h before being treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 4 h. Lysates were immunoblotted with the indicated antibodies.

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