Anti aldoa

Protein extraction and western blot were performed as described previously (Li et al., 2015b). Anti‐RAC1 (1 : 250 dilution) antibody was acquired from Cytoskeleton, Inc. (Denver, CO, USA). Anti‐PKM (1 : 500 dilution), anti‐LDHA (1 : 500 dilution), anti‐ALDOA (1 : 500 dilution), and anti‐HK1 (1 : 500 dilution) antibodies were obtained from Santa Cruz Biotechnology. Anti‐phospho‐AKT (Ser473) (1 : 1000 dilution), anti‐AKT (1 : 1000 dilution), anti‐phospho‐FoxO1 (Thr24)/FoxO3a (Thr32) (1 : 1000 dilution), anti‐FOXO3A (75D8) (1 : 1000 dilution), anti‐phospho‐S6 (Ser240/244) (1 : 1000 dilution), and anti‐S6 (5G10) (1 : 1000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Islets or cells were homogenized in RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 1 mM Na₃VO₄) containing protease inhibitor and phosphatase inhibitor (MCE). Protein lysates were separated by SDS-PAGE on 10% polyacrylamide gels and transferred to PVDF membranes (Millipore). Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies. Blotted membrane was imaged with a LAS-4000 Super CCD Remote Control Science Imaging System (Fuji). Anti-Flag was from Sigma-Aldrich. Anti-tubulin was from Proteintech. Anti-ATP5A, anti-UQCRC2, anti-MTCO1, anti-SDHB, and anti-rabbit/mouse IgG conjugated with HRP were purchased from Cell Signaling Technology. Anti-GCK, anti-GKRP, and anti-ALDOA were from Santa Cruz biotechnology. Anti-phosphoenolpyruvate carboxykinase (PEPCK) was from Abcam. Anti-HSP90 was from Millipore. Anti-acetyllysine was from PTM Biolab. Immunoprecipitation was performed by incubating protein lysates with the indicated antibodies for 2 h and then with protein A/G-agarose beads (Santa Cruz biotechnology) overnight at 4 °C. The immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 10 min. Then, standard western blotting was followed.