Plasma membrane protein extraction kit

Trilobatin, palmitate, DAPI and (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2NBDG) uptake measurement kits were purchased from Sigma (St. Louis, MO, USA). IRS1, p-IRS1 (Ser 612), p-IRS1 (Ser 307), Akt, p-Akt (Ser 473), p-Akt (thr308), Na, K-ATPase, MYH1, MYOD1, and β-actin primary antibodies were bought from Cell Signaling Technology (Danvers, MA, USA). GLUT4 antibody was obtained from Abcam (Cambridge, MA, USA). HRP-conjugated GAPDH primary antibody was purchased from Aksmics (shanghai, China), Specific anti-mouse and anti-rabbit HRP-conjugated second antibodies were obtained from Santa Cruz Biotechnology (Texas, CA, USA). Rat/mouse insulin ELISA kits (EZRMI-13K) and ECL chemiluminescence detection reagent were obtained from Millipore (Billerica, MA, USA). Plasma membrane protein extraction kit, nuclear/cytosolic fractionation kit, RIPA buffer and BCA protein assay kit and other chemicals were purchased from Beyotime (Shanghai, China).

After washing with 1 ml of ice-cold PBS, cells were harvested and lysed with the membrane protein extraction reagent A (Plasma Membrane Protein Extraction Kit, Beyotime, China). After homogenization, the mixture was centrifuged at 700 g for 10 min at 4 °C and the supernatant was further centrifuged at 10,000 g for 30 min at 4 °C. The sediment contains the total membrane protein while the supernatant contains cytosol protein. Further, the pellet was mixed with membrane protein extraction reagent B for 20 min and centrifuged at 10,000 g for 30 min at 4 °C to collect the supernatant.