Detection of MAPK proteins by Western blot analysis

Immortalized cholangiocytes were lysed and protein extracted in 1X Loading Buffer (50mM Tris-HCl pH 6.8, 2% SDS, 10% Glycerol, 0.05% Bromo-Blue, 0.4M 2-mercaptoethanol). Equal amounts of cell lysate per sample were loaded on a 4-20% tris-glycine gel (Invitrogen Corporation, Carlsbad, CA). Gels were transferred overnight to PVDF membranes (GE Healthcare, Pittsburg, PA) and blocked with 5% milk solution. Membranes were stained with a 1:1000 dilution of rabbit anti-mouse p-ERK 1/2 antibody and total p38 antibody as loading control (Cell Signaling Technology Inc. Danvers, MA). Densitometry was utilized to determine fold change between groups.