(A) KRT5 mRNA expression in ovarian cancer cell lines and LP-9 cells. (B) KRT6C mRNA expression in ovarian cancer cell lines and LP-9 cells. Relative expression was normalized to the house keeping gene β-actin using the 2-ΔΔCT method with the same calibrator (OVCAR-3). Data is expressed as the mean fold change ± SEM from 4-11 individual RNA samples obtained from 2-4 independent experiments. (C) Equal amounts protein for the cell lines (40 μg) and ovarian cancer tissue extracts (5 μg) were run on a 4-20% SDS-PAGE gel and immunoblotted with mouse monoclonal K5/6 antibody (1/200, clone D5/16 B4, Dako). K5/6 antibody detected K6 bands at ~52 kDa in all cell line extracts and faint K5 bands at ~56 kDa in cell extracts from OVCAR-5, OV-90 and SKOV-3 cells. Both K5 and K6 were detected in ovarian cancer tissue extracts. White asterisks indicate faint K5 bands detected with K5/6 antibody. (D) Western blotting with rabbit monoclonal K5 antibody (1/5000, clone EPR1600Y, Abcam) antibody using the same protein samples from (C) confirmed K5 expression in ovarian cancer tissue extracts. A mouse monoclonal antibody to β-actin (1/10,000, clone AC-15, Sigma Aldrich A3854) was used as a loading control. Ovarian cancer tissues sections with high and low K5/6 (C) or K5 (D) immunostaining were used as positive controls for the western blots (scale bar = 50 μm).