Western blot and cell surface biotinylation assay

For biotinylation of cell surface GLUT4, cells were washed with ice‐cold PBS three times and incubated with 0.75 mL chilled PBS containing 1.5 mg·mL⁻¹ EZ‐link NHS‐SS‐biotin (Thermo Scientific) for 1 h at 4 °C. After quenching with glycine‐containing PBS (100 mm), cells were lysed in a RIPA buffer (150 mm NaCl, 50 mm Tris/HCl, 5 mm EDTA, 1% Triton X‐100, 0.5% deoxycholate, and 0.1% SDS) containing protease inhibitor mixture. Biotinylated proteins were precipitated by streptavidin‐agarose beads for overnight at 4 °C. The beads were subsequently washed four times with PBS containing 1% Triton X‐100. Biotin‐labeled proteins were eluted in the sample buffer, separated by SDS/PAGE, and transferred to poly(vinylidene difluoride) (PVDF) membranes for western blotting. Blots were developed using enhanced chemiluminescence. Biotinylation experiment was performed 3–4 times with similar results. Primary antibodies used western blots for p‐IGF‐1Rβ^Thr1131/InsRβ^Tyr1146 (1 : 1000 dilution, Cat no. 3021), InsR (1 : 1000 dilution, Cat no. 3025), p‐Akt^Ser473 (1 : 2000 dilution, Cat no. 9271), p‐Akt^Thr308 (1 : 2000 dilution, Cat no. 2965), Akt (1 : 2000 dilution, Cat no. 9272), p‐TBC1D4^Ser588 (1 : 1000 dilution, Cat no. 8730), TBC1D4 (1 : 1000 dilution, Cat no. 2670), and GLUT4 (1 : 2000 dilution, Cat no. 2213) were provided from Cell Signaling Technology (Beverly, MA, USA). Phospho‐WNK1 antibody detecting at threonine58 (T60 for human; 1 : 1000 dilution, Cat no. {"type":"entrez-protein","attrs":{"text":"STJ90776","term_id":"1438914234","term_text":"STJ90776"}}STJ90776) was purchased from St John's Laboratory (London, UK). β‐actin (1 : 10000 dilution, Cat no. ab6276), WNK1(1 : 1000 dilution, Cat no. ab128858), and myc‐HRP (1 : 5000 dilution, Cat no. ab1326) were from Abcam (Cambridge, MA, USA). GAPDH (1 : 3000 dilution, Cat no. sc25778) were purchased from Santa Cruz Biotechnology. Relative protein levels were normalized by internal controls, GAPDH or β‐actin in western blot analysis. Densitometry for western blot bands was analyzed using ImageJ software (version 1.8, National Institutes of Health, Bethesda, MD, USA).