Anti αenac

BEAS-2B cells were seeded in 6-well plates overnight. Cells were pre-incubated with SB431542 (1μM) for 30 min prior to RSV infection at MOI of 0.1 or control medium for 3 hours, 24 hours and 48 hours for assessment of intracellular kinase phosphorylation. To assess changes in total GRα, ENaCα and PLZF expression, budesonide (100nM) was added to the cells following 48 hours RSV infection (MOI 0.1) for the last 2 hours or the last 24 hours. In some experiments, PLZF expression was measured after treatment of the cells with budesonide for 4 hours prior to RSV infection for 48 hours. Rabbit polyclonal antibody (pAb) anti-phospho-ERK1/2 (Thr202/Tyr205) and rabbit monoclonal antibody (mAb) anti-Erk1/2 (Cell Signaling) was used to measure the ERK1/2 activation. Rabbit pAb anti-GRα (Santa Cruz Biotechnology) was used to measure GRα expression. Mouse monoclonal antibody anti-PLZF and goat polyclonal antibody anti-αENaC (Santa Cruz Biotechnology) were used to measure the expression of PLZF and ENaCα. The expression level of GAPDH protein (Rabbit pAb; Abcam, Cambridge, UK) was used as a reference control for normalization to account for variation in protein loading. Western blotting was performed as described [66 (link)]. Band intensities were quantified by densitometry using the image J program (1.48v, National Institute of Health, USA).

Cell lysates were homogenized in buffer, resolved in a 10% SDS-PAGE and analyzed by immunoblotting with specific antibodies (Ab); anti-α1-Na,K-ATPase (1μg/ml, Cat# ab7671, Abcam), anti-β-actin (1:1000, Cat# 4970, Cell Signaling Technologies, inc.), anti-angiotensin receptor type 1 (1:500, Cat# sc-1173, Santa Cruz Biotechnologies, inc), anti-αENaC (1:1000, Cat# sc-21012, Santa Cruz Biotechnologies, inc) and anti-αtubulin (1:3000, Cat# ab126165, Abcam). ECL kit (Thermo Scientific, USA) was used to develop the image.