Irdye 680 lt conjugated polyclonal donkey anti mouse igg

Western blot analysis was performed as previously described [17] (link). After overnight serum starvation, primary lung fibroblasts ± nicotine (50 µg/ml) were cultured in complete serum-free media for 24–72 hours as noted in the figure legends. Cytoplasmic and nuclear protein fractions were isolated using the Nuclear Extract Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. Primary antibodies employed included polyclonal rabbit anti-p65 (1∶1000, Cell-Signaling), polyclonal rabbit anti-phospho-p65 Ser536 (1∶500, Cell Signaling), monoclonal mouse anti β-tubulin (1∶500, Millipore), polyclonal rabbit anti-histone (1∶2000, Cell Signaling), and polyclonal rabbit anti-GAPDH (1∶10,000, Sigma). Secondary antibodies used were IRDye 680 LT conjugated polyclonal donkey anti-mouse IgG (1∶20,000, LI-COR Biosciences) and IRDye 800 CW conjugated polyclonal goat anti-rabbit IgG (1∶20,000, LI-COR Biosciences). Quantification of protein expression was performed by measuring integrated intensity using the Odyssey Infrared Imaging System (LI-COR Biosciences) and measuring densitometry using ImageJ software (National Institutes of Health) and then normalized to the appropriate loading control. Results are reported as fold change compared to untreated conditions.