Hrp conjugated secondary goat anti mouse and goat anti rabbit antibodies

Lung tissue slices were immunoblotted for total protein analysis as described previously (12 (link)). Protein concentration was determined from native cell lysates using a bicinchoninic acid protein assay (Thermo Fisher Scientific, Lafayette, CO). Lysates were denatured, and equal amounts of protein were subjected to SDS-PAGE followed by immunoblotting. Blots were probed with rabbit anti-phospho P65 (Cell Signaling 3033, 1:3000), rabbit anti P65 (Cell Signaling 3034, 1:2000), rabbit anti-VCAM (Abcam ab134047, 1:1000) and mouse anti-GAPDH (Abcam ab135247, 1:2000). HRP-conjugated secondary goat anti-mouse and goat anti-rabbit antibodies (Invitrogen) were detected by enhanced chemiluminescence (SuperSignal™ West Femto, ThermoFisher).

For total protein analysis, tissue was immunoblotted as described previously [69 (link)]. Briefly, tissues were frozen in liquid nitrogen, and then ground into a fine powder with a mortar and pestle. Tissue powder was then resuspended in RIPA buffer supplemented with protease inhibitor cocktail (Complete Mini, Roche, Bath, UK) for 1 hour on ice. Tissue solutions were homogenized for 30 seconds and subsequently centrifuged at 10,000 rpm for 10 minutes. The supernatant was removed and protein concentration was determined from native cell lysates using a bicinchoninic acid protein assay (Thermo Fisher Scientific, Lafayette, CO) with bovine serum albumin as a standard. Lysates were denatured, and equal amounts of protein (10 µg for vimentin) were subjected to SDS-PAGE followed by immunoblotting as described previously [69 (link)]. We used HRP-conjugated secondary goat anti-mouse and goat anti-rabbit antibodies (Invitrogen) detected by enhanced chemiluminescence.