6 cm pvdf membranes

For protein expression analyses with western blots, PBL were first lysed in lysis buffer (9 M Urea, 2 M Thiourea, 65 mM Dithioerythritol, 4% CHAPS). Proteins were then separated by SDS-PAGE on 8% gels (7 μg protein/slot) and blotted semidry onto 8.5 × 6 cm PVDF membranes (GE Healthcare, Freiburg, Germany) and blocked with 4% BSA (1 h). Blots were incubated overnight with respective primary antibodies: rabbit anti-pSTAT1 Tyr701, rabbit anti-pSTAT3 Tyr705 (Cell Signaling, Darmstadt, Germany, 1:500) or mouse anti-beta actin (Sigma, Taufkirchen, Germany, 1:5000). As secondary antibodies, either HRP-coupled goat anti-rabbit IgG (H+L) antibody (Cell Signaling, Darmstadt, Germany, 1:5000) or goat anti-mouse IgG (H+L) antibody (Sigma-Aldrich, Taufkirchen, Germany, 1:5000) were used (1 h). Signals were detected by enhanced chemiluminescence on X-ray film (SUPER-2000G ortho, Fuji; Christiansen, Planegg, Germany). Films were scanned on a transmission scanner and densitometric quantification of Western blot signals was performed using ImageJ software (open source: http://imagej.nih.gov/ij/). Abundances of pSTAT1 and pSTAT3 were subsequently normalized to beta actin.

DC pellets were lysed in RIPA lysis buffer (Sigma-Aldrich) with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). Proteins were then separated with SDS-PAGE 4–20% gel (20 μg protein/slot; Precast Gels, Genscript, Piscataway, NJ) and transferred onto 8.5 × 6 cm PVDF membranes (GE Healthcare, Freiburg, Germany) and blocked with 5% w/v BSA (0.5 h). Membranes were incubated overnight with primary Abs: rabbit anti-p-4E-BP1 (Thr37/46), rabbit anti-p-p70S6K (Thr389/412), rabbit anti-Pparγ, rabbit anti-p-NFκβ p65 (Cell Signaling Technology; 1:1000), rabbit anti-CD36 (Abcam, Cambridge, MA, 1:1000) or mouse anti-β-actin (Sigma-Aldrich; 1:1:2000). HRP-conjugated goat anti-rabbit IgG (H±L) secondary Ab (Cell Signaling Technology; 1:5000) was used (1 h). Signals were detected by Western HRP Substrate on ChemiDoc™MP Imaging System (Sigma-Aldrich; Bio-Rad, Hercules). Densitometric quantification of Western blot signals was performed using Image Lab software (open source: http://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z). All proteins were subsequently normalized to β-actin.