Figure 1.

Western blot and confocal analysis of MUC4 and HER2 in SKOV3 cells. (A) Western blot analysis of MUC4 expression and its derived sub lines SKOV3 Vec (empty vector p-SecTaq) and SKOV3 MUC4. A total of 20 μg protein from cell extracts was resolved by electrophoresis on a 2% SDS-agarose gel for MUC4 and 10% SDS-PAGE for HER2, transferred to polyvinylidene difluoride membrane, and incubated with anti-MUC4 monoclonal antibody. The membrane was then probed with horseradish peroxidase-labeled goat anti-mouse immunoglobulin. The signal was detected using an electrochemiluminescence reagent kit. MUC4 mucin is a high molecular weight glycoprotein and the predicted size of the mini MUC4 protein is 320 kda. β-actin served as a loading control. (B) Localization of MUC4 and HER2 by confocal microscopy in both the derived cells. Cells were grown at low density on sterilized cover slips, washed, and fixed in ice-cold methanol at -20°C. After blocking in 10% goat serum, cells were incubated with the anti-MUC4 mouse monoclonal and anti-HER2 rabbit polyclonal antibodies, washed, and followed by secondary incubation with FITC-conjugated goat anti-mouse IgG and anti rabbit PI used for nuclear staining (Scale bar-20 μm).