Quantitative Protein Expression in HCE Cells

Western blot analysis was performed on SkinEthic^TM HCE cells as previously described [73 (link)]. Protein concentration was estimated by the Bio-Rad protein assay using bovine serum albumin (BSA) as standard. Samples were heated at 95 °C for 5 min, and the same amounts of protein were separated on 12% SDS-PAGE gel and blotted to PVDF membrane (Immobilon-P). Membranes were incubated overnight (O/N) at 4 °C with the following primary antibodies: anti-occludin (1:500, Cat#91131, Cell signaling, Danvers, MA, USA) or anti-mucin 1 (1:500; sc-7313, Santa Cruz Biotechnology, Santa Cruz, CA, USA). To ascertain that the blots were loaded with equal amounts of proteins, the membranes were also incubated in the presence of the antibody against β-actin protein (1:1000; sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Results are expressed as % of the control.