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Published in final edited form as: Cell Signal. 2013 Feb 14;25(5):1156–1165. doi: 10.1016/j.cellsig.2013.02.005

(A) Immortalized wild-type (RKIP+/+) and RKIP^−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1) or control siRNA (siCon). After 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot. (B) RKIP was stably depleted in WT and Raf-1−/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, and Raf-1 were detected by western blot. (C) Immortalized wild-type and RKIP−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1), B-Raf siRNA (siB-Raf), double B-Raf and Raf-1 siRNA (siRaf-1+siB-Raf) or control siRNA (siCon); after 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP, Raf-1 and B-Raf were detected by western blot. *, p<0.03. (D) Raf-1 was transiently knockdown by electroporation of siRNAs targeting Raf-1 in RKIP depleted B-Raf −/− MEFs. After 24 hours at 37°C, cells were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) at 37°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot.* p<0.01. (A-B-C-D) Western blots show a representative experiment. The bar charts represent the densitometric quantification of pERK/ERK ratios from two independent experiments; error bars represent range. UT: untreated.

(A) Immortalized wild-type (RKIP+/+) and RKIP^−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1) or control siRNA (siCon). After 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot. (B) RKIP was stably depleted in WT and Raf-1−/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, and Raf-1 were detected by western blot. (C) Immortalized wild-type and RKIP−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1), B-Raf siRNA (siB-Raf), double B-Raf and Raf-1 siRNA (siRaf-1+siB-Raf) or control siRNA (siCon); after 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP, Raf-1 and B-Raf were detected by western blot. *, p<0.03. (D) Raf-1 was transiently knockdown by electroporation of siRNAs targeting Raf-1 in RKIP depleted B-Raf −/− MEFs. After 24 hours at 37°C, cells were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) at 37°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot.* p<0.01. (A-B-C-D) Western blots show a representative experiment. The bar charts represent the densitometric quantification of pERK/ERK ratios from two independent experiments; error bars represent range. UT: untreated.