Mca2748

We evaluated translocation of Cd36 and Glut4 to the cell surface as previously described^{26 (link)}. We serum-starved 3T3-L1 adipocytes for 12 h and treated with rMfge8 or RGE construct (10 µg ml⁻¹), insulin (1 µM), wortmannin (100 nM) or Pkc-ζ or control inhibitor (5 µM) for 20 min. We then fixed cells with 3% paraformaldehyde and blocked for 1 h with blocking buffer (PBS, 0.05% Tween 20, 1% BSA, 5% goat serum). We then incubated cells with rat antibody against mouse Cd36 (Abd Serotec, MCA2748) or Glut4 (N-20, Santa Cruz, D2613) at a concentration of 1:500, followed by a secondary horseradish peroxidase–conjugated antibody (Santa Cruz) at a concentration of 1:1,000, followed with 3,3′,5,5′-Tetramethylbenzidine Liquid Substrate System (TMB, Sigma) at room temperature, after which we terminated the reaction with 1 N NaOH and read with a plate reader (Molecular Devices, SpectraMax M2) at 450 nm. We subsequently rinsed cells with PBS and incubated with wheat germ agglutinin Alexa Fluor 680 conjugate (WGA Alexa-680, Invitrogen) for 30 min, washed again with PBS and used Odyssey infrared imager (Licor) to read the signal at 700 nm. We subtracted background signals from the infrared signal as well as from controls read by TMB immunostaining with primary antibodies omitted from the raw data.