Trans blot semi dry electrophoretic transfer system

Lung cells or MDCK cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA) containing Protease Inhibitor Cocktail (Sigma‐Aldrich). An aliquot of the lysate (50 μg for lung cells or 20 μg for MDCK cells) was separated in a 10% Bis‐Tris gel (Life Technologies) and transferred onto a polyvinylidene difluoride membrane (PVDF, Life Technologies) by using Trans‐Blot semi‐dry electrophoretic transfer system (Bio‐Rad Laboratories). After treatment with PVDF Blocking Reagent (TOYOBO, Osaka, Japan), the membrane was probed with a primary monoclonal antibody against influenza A virus M2 protein (clone 14C2, 1:500, Abcam) or β‐actin (clone AC‐15, 1:2000, Sigma‐Aldrich) and a horseradish peroxidase (HRP)‐conjugated secondary antibody against the primary antibody (1:500, Santa Cruz Biotechnology). The signals were visualized using an ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ).

Samples were prepared in radioimmunoprecipitation assay (RIPA) buffer, boiled, and separated by SDS-PAGE on 10% polyacrylamide gels, followed by transfer to Immobilon-P membranes (Millipore) using the Trans-Blot semi-dry electrophoretic transfer system (Bio-Rad). Membranes were blocked for 1 h at RT with 5% skim milk in TBS containing 0.1% Tween 20 (TBST) and then incubated at 4°C overnight with primary antibodies specific for α-SMA (Sigma-Aldrich), tubulin (Cell Signaling), phospho-Smad2 (Cell Signaling), phospho-Smad3 (Abcam), Smad2 (Cell Signaling), Smad3 (Abcam), integrin beta 3 (Abcam), integrin beta 5 (Abcam), integrin beta 6 (R&D systems), and β-actin (Cell Signaling) (all diluted in 2% skim milk in TBST). Membranes were washed and then incubated with HRP-conjugated goat anti-mouse, goat anti-rabbit, or donkey anti-goat IgG secondary antibodies (Jackson ImmunoResearch, Suffolk, UK) diluted in 2% skim milk in TBST. Blots were developed with the SuperSignal West Pico chemiluminescent substrate system (Thermo Fisher Scientific).