Figure 3

The mucin MUC4 interacts with HER2 and induces the expression of HER2. (A) Estimation of HER2 mRNA levels in MUC4-transfected SKOV3 ovarian cancer cells by real-time RT–PCR. A total of 2 μg of RNA was reverse-transcribed and diluted to a total volume of 100 μl (5 × dilution). A total of 1 μl of this cDNA was used in each reaction in a total reaction volume of 10 μl. Each reaction was plated in triplicate. The results are expressed as the fold changes of HER2 in MUC4-expressed SKOV3 cells relative to that in the vector-transfected cells. No significant change was observed in HER2 transcript levels compared with empty vector-transfected cells (P-values by applying the t-test: 0.889). (B) Western blot analysis for HER2 expression and HER2 tyrosine phosphorylation in SKOV3-derived cell lines. A total of 20 μg of protein from the cell lysate was resolved by SDS–PAGE, transferred to a PVDF membrane and probed with antibodies against MUC4, HER2, phosphor-Tyr¹²⁴⁸ HER2 and β-actin. SKOV3 cells transfected by MUC4 showed an increased expression and tyrosine phosphorylation of HER2 at the Tyr¹²⁴⁸ residue. No phosphorylation of HER2 was seen in the SKOV3-vector control. (C) Reciprocal co-immunoprecipitation analysis to show the interactions between MUC4 and HER2. Lysates from the MUC4-overexpressing SKOV3 cell lines were utilised for immunoprecipitation with MUC4 and HER2 antibodies. The immunoprecipitates were electrophoretically resolved on 2% SDS-agarose (for MUC4) and 10% polyacrylamide gel (for HER2), and immunoblotted with anti-MUC4 or anti-HER2 antibodies. The isotype antibodies were used as immunoprecipitation controls. (D) Co-localisation of MUC4 and HER2 in SKOV3 MUC4-transfected cells. Cells were grown at low density on sterilised cover slips, washed and fixed in ice-cold methanol at −20 °C. After blocking with 10% goat serum, cells were incubated with the anti-MUC4 mouse monoclonal antibody and anti-HER2 polyclonal antibody followed by incubation with FITC-conjugated goat anti-mouse IgG (× 630 original magnification). The green staining shows MUC4 expression, red staining shows HER2 expression and the yellow staining shows the co-localisation.