Gr pa1 511a

Western blotting was carried out as described previously (39 (link)). Following cell lysis, proteins were size-fractionated in SDS-PAGE gels. Following electrophoresis, the proteins were transferred to a nitrocellulose membrane followed by blocking and probing with primary antibodies against TLR2 (12276, Cell Signaling and ab108998, Abcam), GFP (2555, Cell Signaling), IκBα (sc-371, SantaCruz), Ser32/Ser36 phosphorylated-IκBα (9246, Cell Signaling), p65 (sc-8008, SantaCruz), Ser536 phosphorylated-p65 (3036, Cell Signaling), GR (PA1-511A, Thermo Fisher Scientific), MAPKAPK2 (3042, Cell Signaling), Thr334 phosphorylated-MAPKAPK2 (3041, Cell Signaling), total p38 (9212, Cell Signaling), Thr180/Tyr182 phosphorylated-p38 (9211, Cell Signaling), p38α (9218, Cell Signaling), p38β (2339, Cell Signaling), or GAPDH (MCA4739, Bio-Rad), overnight at 4 ºC. Membranes were washed in Tris Buffer Saline-Tween 20 followed by incubation with 1:5000 or 1:10,000 dilution of the respective rabbit or mouse horseradish peroxidase-conjugated secondary immunoglobulin (Jackson ImmunoResearch) at room temperature. Membranes were washed 4 × 5 min prior to detection of immune complexes by enhanced chemiluminescence (Bio-Rad). Images were acquired using a ChemiDoc Touch imaging system (Bio-Rad), and densitometric analysis was performed using ImageLab software (Bio-Rad).