Irdye 680 conjugated goat polyclonal anti rabbit igg h l

Samples of protein (50 µg) extracted from the lung (n = 4–5 from each group) were applied to polyacrylamide gel/SDS 10% and then transferred to nitrocellulose membranes. Membranes were incubated overnight with different primary antibodies: GATA3 (Rabbit anti-GATA3, 1:500, Abcam Labs, Cambridge, UK); total Ikβα (rabbit anti-t-IkBα; 1:1,000; Cell Signaling, MA, USA) or phosphorylated IkBα (rabbit anti-p-Ikβα; 1:500; Cell Signaling, MA, USA); total ERK1/2 (rabbit anti-t-ERK1/2; 1:1,000; Cell Signaling; MA, USA), or phosphorylated ERK1/2 (rabbit anti-p-ERK1/2, 1:500; Cell Signaling; MA, USA). Next, membranes were incubated with fluorescent secondary antibody [IRDye ^® 680 conjugated goat (polyclonal) anti-Rabbit IgG (H + L) diluted to 1:10,000 (Li-COR Biosciences, NE, USA)]. Levels were normalized to GAPDH levels in the same sample. Staining was visualized and quantified in a Li-COR Odyssey Scanner (Biosciences, NE, USA).

Western blot was performed essentially the same as previously described54 (link). Briefly, cells were lysed in RIPA buffer (Cell Signaling Technology, Boston, MA, USA) containing protease and phosphatase inhibitors (Roche, Penzberg, Germany). The concentrations of protein in the lysates were determined with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s protocol. After electrophoresis on 10% SDS-PAGE Bio-Rad minigels, the aliquots proteins were transferred to nitrocellulose filter membrane (Whatman/GE Healthcare life sciences, Marlborough, MA, USA). The blots were blocked for 1 hour at room temperature in 5% skim milk in TBST, and incubated overnight with rabbit anti-Influenza A Virus Nucleoprotein antibody (GeneTex, Irvine, CA, USA) and mouse anti-β actin monoclonal antibody (ZSGB-BIO, Beijing, China) at a dilution of 1:10,00 as primary antibodies. IRDye 680-conjugated goat polyclonal anti-rabbit IgG (H+L) and IRDye 800-conjugated goat polyclonal anti-mouse IgG (H+L) (both from LiCor, Lincoln, NE, USA) were used as secondary antibodies at a dilution of 1:10,000. The blot was examined and photographed with a Odyssey imaging systems (LiCor, Lincoln, NE, USA).