Journal: Life Metabolism
Article Title: Glucose-6-phosphate dehydrogenase regulates mitophagy by maintaining PINK1 stability
doi: 10.1093/lifemeta/loae040
Figure Lengend Snippet: G6PD interacts with the mitophagy machinery. (a) Mitochondrial G6PD levels detected by immunoblotting analysis. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 1 h or 2 h. Cells were subjected to fractionation by sucrose gradient centrifugation and immunoblotted with the indicated antibodies. Sample loading was standardized to the whole cell lysate. GAPDH, cytosolic marker; TIM23, mitochondrial marker. WCL, whole cell lysate; Cyto, cytosol; Mito, mitochondria. (b) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and TOM20 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (c) Visualization of G6PD mitochondrial localization by proteinase K protection assay. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and subjected to fractionation by sucrose gradient centrifugation. The mitochondrial fraction was divided and treated with the indicated concentrations of digitonin and proteinase K. Samples were then subjected to immunoblotting analysis. (d) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and PINK1 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (e) Immunoprecipitation of overexpressed myc-PINK1 with endogenous G6PD. HeLa 3+ cells were transfected with myc-PINK1 for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and lysed in IP lysis buffer. Lysates were subjected to IP with agarose-conjugated G6PD antibody and blotted with the indicated antibodies. (f) In vitro pull-down assay between G6PD and PINK1. HeLa mCherry-Parkin cells were transfected with FLAG-tagged G6PD. FLAG-G6PD protein was isolated using FLAG M2 beads and incubated with recombinant human PINK1 protein. Samples were subjected to SDS-PAGE and western blotting analysis to visualize pull-down of PINK1.
Article Snippet: The following primary antibodies were used: Cell Signaling Technology: AMPKα (#5832), GFP (#2956), SAPK/JNK (#9252), K48-linkage Specific Polyubiquitin (#8081), Mitofusin-1 (#14739), Mitofusin-2 (#11925), phospho-AMPKα (Thr172) (#2535), phospho-SAPK/JNK (Thr183/Tyr185) (#4668), phospho-Ubiquitin (Ser65) (#62802), Parkin (#4211), PINK1 (#6946), and TOM20 (#42406); Santa Cruz Biotechnology: G6PD (sc-373886) and HA tag (sc-7392); Sigma-Aldrich: FLAG® M2 (F1804), α-Tubulin (T6199), and β-Actin (A5441); Abcam: GAPDH (ab8245), mCherry (ab167453), and MTCO2 (COXII) (ab110258); BD Transduction Laboratories: TIM23 (611222); Proteintech: VDAC1/2 (10866-1-AP).
Techniques: Western Blot, Fractionation, Gradient Centrifugation, Marker, Immunoprecipitation, Transfection, Lysis, In Vitro, Pull Down Assay, Isolation, Incubation, Recombinant, SDS Page