Ab87230

Antibodies used in this study were principally purchased from Santa Cruz Biotechnology (G6PD (G-12): SC-373886 (dilution 1:1000), 6PGD (G-2): SC-398977 (dilution 1:1000), His (HIS.H8): SC-57598 (dilution 1:1000), Enolase (H-300): SC-15343 (dilution 1:1000)), Cell Signaling Technology (beta actin (8H10D10): 3700 S (dilution 1:1000)), Everest Biotech (G6PD: EB07841 (dilution 1:1000)), Advance Immunochemical (GAPDH (6C5): 6C5 (dilution 1:2000)), and Abcam (G6PD: AB87230 (dilution 1:1000), band 3 (EPR1426): AB108414 (dilution 1:1000)). TALON Superflow (28-9575-02) and bovine thrombin (91-030) were purchased from GE Healthcare and BioPharma for protein purification. Pfu Turbo polymerase (600252) used for site-directed mutagenesis was purchased from Agilent Technologies. Chymotrypsin was purchased from Promega (V1061). Cell counting kit-8 (CK04) and glutathione quantification kit (T419) were purchased from Dojindo. Cycloheximide (C7698) and chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA, C6827) were purchased from Sigma and Thermo Fisher Scientific, respectively. Glutathione assay kit (703002) used for blood assays was purchased from Cayman Chemical.

The protein samples were separated by SDS-PAGE and then transferred to a nitrocellulose membrane (PVDF Western Blotting Membranes, Roche, Basel, Switzerland) by semidry immunoblotting (Bio-Rad). The presence of proteins on the membrane was confirmed by Ponceau S (Sigma-Aldrich) labeling. After blocking with a PBST buffer (PBS, 0.1% Tween 20) containing 3% nonfat milk, the following primary antibodies were used: anti-yeast alcohol dehydrogenase (1:5000, ab34680, Abcam, Cambridge, UK), anti-yeast aldehyde dehydrogenase (1:5000, ab182893, Abcam), anti-glucose-6-phosphate dehydrogenase (1:2000, ab87230, Abcam), anti-6-phosphogluconate dehydrogenase (1:2000, ab125863, Abcam), and anti-β-actin (1:5000, MA5-15739, Thermo Fisher Scientific). The respective proteins were detected after incubation with the horseradish peroxidase-conjugated secondary antibodies (1:10,000, 111,035,003, Jackson ImmunoResearch or 1:10,000, AP160P, Millipore, Merck, Rahway, NJ, USA) with a SuperSignal West PICO Chemiluminescent Substrate (Pierce Biotechnology, Waltham, MA, USA), according to the manufacturer’s protocol. The images were captured using an Imaging Systems c300 (Azure Biosystems, Inc., Dublin, CA, USA).