β actin rabbit

We passage BEAS-2B cells into 6-well plates. Radioimmunoprecipitation assay (RIPA) lysate (Invitrogen, Carlsbad, CA, USA) is used to lyse cells and extract proteins. The bicinchoninic acid (BCA) method (Beyotime, Shanghai, China) was used to detect protein concentration. We configured 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) gel according to the instructions. The protein (30 μg) was then added to each well of the electrophoresis gel for SDS-PAGE. We then transferred the protein band to the polyvinylidene fluoride (PVDF) membrane through transfer (Roche, Basel, Switzerland). After washing the PVDF membrane with phosphate buffered saline-tween (PBST), we blocked it with 5% skim milk and incubated it with primary antibody dilution (MUC1, rabbit, 1:3000, Abcam, Cambridge, MA, USA; TLR4, 1:2000, rabbit, Abcam; NF-κB p65, 1:3000, rabbit, Abcam; β-actin, rabbit, 1:3000, Abcam) for 4°C overnight. We then incubated the PVDF membrane for 2 h at room temperature using secondary antibody dilution (Goat anti-rabbit, 1:3000, Abcam). Finally, we detected protein bands by electro-chemi-luminescence (ECL).