Anti rabbit horse radish peroxidase conjugated secondary antibodies

Mouse anti-Fbxo7 (Santa Cruz, sc-271763), mouse anti-GAPDH (Santa Cruz, sc-365062), mouse anti-Flag (Sigma, F1804), mouse anti-β-Actin (Sigma, A2228), mouse anti-Myc (Santa Cruz, sc-40), mouse anti-V5 (Bio-Rad, MCA1360GA), mouse anti-Ubiquitin (Abcam, ab134953), rabbit anti-Bag2 (Abcam, ab79406), mouse anti-HA (Abcam, ab130275), anti-mouse horseradish peroxidase-conjugated secondary antibody (Rockland, 18-8817-31) and anti-rabbit horseradish peroxidase-conjugated secondary antibody (Rockland, 18-8816-31) for IP Western blots, anti-mouse horse radish peroxidase-conjugated secondary antibodies (Millipore, AP187P) and anti-rabbit horse radish peroxidase-conjugated secondary antibodies (Millipore, 12-348) for regular Western blots. All primary antibodies were used at 1:5000 dilution, except anti- β-Actin, which was diluted 1:50,000 for Western blot analyses. The secondary antibodies for IP were diluted 1:5000. The secondary antibodies for Western blots were diluted 1:10,000.

Homogenised lung tissue was preserved in a protease-inhibitor solution (Complete Mini; Roche). Western blot analyses were performed for PPAR-γ, inhibitor-κβ (Iκβ) and NF-κβ p65 using 20 μg protein. The PPAR-γ specimens were separated by electrophoresis in a 10% Bis-Tris gel, while the NF-κβ p65 and Iκβ specimens were separated by electrophoresis in a 3–8% Tris-acetate gel. Following electrophoresis, the proteins were transferred to a nitrocellulose membrane. Primary antibodies against β-actin, PPAR-γ, phospho-PPAR-γ (Cell Signaling Technology, Inc., Danvers, MA, USA), NF-κβ p65 and Iκβ (R&D Systems, Minneapolis, MN, USA) were applied overnight at 4°C in Tris-buffered saline, which was followed by the addition of anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Millipore, Billerica, MA, USA) for 1 h at room temperature (RT). Chemiluminescence detection was performed with the Western Lightning detection reagent (Perkin-Elmer, Waltham, MA, USA).