FIGURE 5.

The importance of RFFL in EGF-CRAF and activated BRAF-stimulated sustained PKC HM phosphorylation and cell migration. A, elevation of RFFL mRNA levels by Raf expression. HEK293T cells were transfected with the Raf cDNAs, and the relative levels of RFFL mRNA were determined by quantitative RT-PCR the next day. Data are presented as mean ± S.D. *, p < 0.05 versus LacZ, Student's t test. B–D, CRAF is required for EGF-induced RFFL expression and phosphorylation of PKCδ and MARCKS. MEFs were transfected with the Raf siRNAs for 2 days and stimulated with EGF for 2 h before quantitative RT-PCT (B and C) or Western blot analysis (D). The relative mRNA levels of Rffl (B and C) were determined by quantitative RT-PCR. Data are presented as mean ± S.D. *, p < 0.05 versus Vc, Student's t test. siCTR, siRNA control; Vc, vehicle control. E and F, important roles of RFFL in EGF-induced PKCδ HM and MARCKS phosphorylation and cell migration. MEFs were transfected with RFFL siRNA for 2 days and analyzed by Western blotting (E) or scratch migration assay (F). The Western blot analysis was quantified from three blots. Quantitative data are presented as mean ± S.D. *, p < 0.05, Student's t test. G and H, important roles of RFFL in PKCδ HM and MARCKS phosphorylation and cell migration of tumor cells harboring activated BRAF. Cells were transfected with RFFL siRNA for 2 days and analyzed by a transwell migration assay (G) or Western blot analysis (H). The data in G are presented as mean ± S.D. *, p < 0.01; **, p < 0.05; Student's t test).