Monoclonal mouse anti human β actin

Cells were seeded in six-well plates and treated per the experimental design. Total protein was obtained after lysis, and the cleared lysates were denatured by boiling for 10 min with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer. The proteins were separated by electrophoresis with Tris-glycine gels and carefully transferred onto polyvinylidene difluoride (PVDF) membranes in the dark. Then, the PVDF membranes were blocked with 5% evaporated milk for 1 h and incubated overnight with primary antibodies against BMP2 (Abcam, USA) and Smad7 (Santa Cruz, USA). After washing, the membranes were probed with a fluorescently labeled secondary antibodiesy. The immune-reactive signals were detected using a Bio-Rad machine. In addition, the membranes were incubated with a monoclonal mouse anti-human β-actin (Abcam, USA) antibody as a loading control. The relative band intensity was measured using ImageJ analysis software [35 (link), 36 (link)].

After exposure to normocapnia (5% CO₂) or hypercapnia (20% CO₂) for 24 h, differentiated NHBE cells were lysed in RIPA buffer (Santa Cruz Biotechnology) supplemented with PMSF, sodium orthovanadate and protease inhibitor cocktail. Lysate proteins (30 μg/well) were resolved by SDS/PAGE 4–20% gradient gels and transferred to nitrocellulose (Bio-Rad Laboratories). Membranes were probed with polyclonal rabbit anti-human TLR4 (H-80) antibody followed by HRP-conjugated anti-rabbit secondary antibody (Pierce). Blots were stripped and re-probed with monoclonal mouse anti-human β-actin (Abcam) followed by HRP-conjugated anti-mouse secondary antibody (Pierce) to confirm the equal loading. The signals were detected using enhanced chemiluminescence SuperSignal West Dura Substrate kit (Pierce). TLR4/βactin ratios were assessed using ImageJ^{79 (link)}.