PAF receptor is required for EGFR transactivation in ovarian cancer cells. (A) Lysates from SKOV-3, CAOV-3, OVCA433, ES-2 and RMUG-L cells were subjected to SDS-PAGE, followed by immunoblotting for PAFR. Actin was used as loading control. (B) The relevant mRNA levels of PAFR in these five cell lines were tested by quantitative real-time PCR, and the data were normalized to 18S rRNA. For the immunofluorescence staining of phosphorylated EGFR in SKOV-3 (C) and RMUG-L (D) cells, after 10 min of incubation without any drug or with 100 nM PAF, the cells were labeled with a polyclonal antibody to phosphor-EGFR overnight, followed by incubation with fluorescent secondary antibody to phosphor-EGFR for 1 h and staining with DAPI for 10 min. (E) SKOV-3 cells were pretreated for 1 h with increasing concentrations of the PAFR antagonist WEB2086. The cells were then stimulated with PAF (100 nM) for 5 min before they were harvested and subjected to Western blotting. The data shown are representative of at least three independent experiments. Data were analyzed by Student’st-test. * p < 0.05.