Mouse anti fabp4

Cells and tissues were collected and lysed in 40 μl RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate) supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Protein concentration was determined with the BCA Protein Assay (ThermoFisher). Protein extracts (30–40 μg) were mixed with Novex Sample Buffer and Reducing Agent (Life Technologies), resolved by SDS-PAGE in 12%, 10% or 4–12% gradient gels and blotted onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked for 1 h with 5% non-fat dry milk (Bio-Rad) or 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), and then incubated overnight at 4 °C with the following primary antibodies: mouse anti-β-actin (1:3000; Sigma-Aldrich); mouse anti-FABP4 (1:500, Santa Cruz Biotechnology); rabbit anti-adiponectin (1:1000; Thermo-Fisher Scientific); rabbit anti-Emilin-2 (1:1000 [45 (link)], antibody specificity shown in Additional file 1: Fig. S2F). After three washes for 10 min in TBS-T, membranes were incubated for 1 h with goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:1000; Bethyl). After three further washes for 10 min in TBS-T, bands were detected with LiteAblot Extend chemiluminescent substrate (Euroclone), using a ImageQuantLAS 4000 digital imager (GE Healthcare).