Glut4 antibody

Cytoplasmic and cytomembrane proteins were extracted from the hippocampi of mice. Protein content was quantified through the Bradford method. Proteins (50 μg) were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel for electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blotted with Glut4 antibody (Santa Cruz, CA, USA; 1:500), GAPDH antibody (ImmunoWay, TX, USA; 1:500), and Na⁺/K⁺-ATPase antibody (Santa Cruz, CA, USA; 1:500) and incubated with horseradish peroxidase-conjugated second antibody (1:5,000). Immunoreactive bands were visualized using enhanced chemiluminescence.

CYC31 was synthesized according to its natural structure in our lab [22 (link)]. Recombinant human PTP1B protein was also purified in our lab [18 (link)]. I-OMe AG538, insulin, palmitate, MTT, and fatty acid-free BSA were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2-NBDG was obtained from Invitrogen (Carlsbad, CA, USA). DMEM, fetal bovine serum, and horse serum were bought from Hyclone (South Logan, UT). Penicillin-streptomycin solutions were purchased from Millipore (Billerica, MA, USA). ECL Western Blotting Substrates were bought from Bio-Rad (Hercules, CA, USA). pIRS1 (Tyr632) antibody and GLUT4 antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pAkt (Ser473) antibody, pIRβ (Tyr1146) antibody, insulin Receptor β antibody, and IRS1 antibody were bought from Cell Signaling Technology (Danvers, MA, USA). β-actin antibody was obtained from Abcam (Cambridge, MA, USA). RNAiso Plus, PrimerScript^TM RT reagent Kit, and SYBR Premix ExTaq^TM Ⅱ were obtained from Takara (Dalian, Liaoning, China).