Purification and Characterization of Recombinant Human Resistin

We produced hResistin in a eukaryotic cell line [6 (link)]. Briefly, the pcDNA5/FRT/TOPO TA vector containing C-terminal FLAG-tagged hResistin cDNA was integrated into the genome of the Flp-In™ T-REx™ 293 cell line in a Flp recombinase-dependent manner (Invitrogen, Carlsbad, CA). Production of recombinant (r) hResistin in T-REx 293 cells was induced by 1 μg/mL tetracycline. hResistin protein then was purified from the cell culture medium by anti-FLAG M2 antibody agarose (Sigma, St. Louis, MO) column chromatography. To determine the purity of eluted hResistin proteins, SDS-PAGE were employed using 4–20% Criterio Tris-HCl protein gel (#3450033, Bio-Rad, Hercules, CA) and Coomassie (#1610436, Bio-Rad) staining. We then stimulated 3T3-L1 embryonic fibroblasts (ATCC® CL-173™, ATCC, Manassas, VA) with hResistin at different doses for 10 minutes and lysed the cells with Laemmli sample buffer. We analyzed the cell lysates by immunoblotting with anti-phospho-Akt [2 (link), 18 (link), 20 (link)] (#4060, Cell Signaling Technology, Danvers, MA) and anti-GAPDH (G8795, Sigma-Aldrich) to determine the activity of the purified hResistin. Western blot analysis was performed as previously described [18 (link)]. The Trans-Blot Turbo Nitrocellulose Transfer Kit (#1704271, Bia-Rad) were used and protein bands were visualized by chemiluminescence (ECL; RPN2106, GE Healthcare, Marlborough, MA).