Figure 10.

Effect of RAF RNAi on the SLIT2 overexpression-induced inhibition of the phosphorylation levels of MEK1/2 and ERK1/2. The granulosa cells were transfected with B-RAF and/or RAF1 specific siRNA, scrambled siRNA (negative control) or no siRNA (blank control). (A) The expression of the B-RAF and RAF1 genes before and after the GCs were transfected with specific siRNA for 48 h was examined by qRT-PCR. The mRNA expression was normalized to that of the 18S rRNA gene; the values on the bar graphs represent the mean ± SEM of 10 hens (n = 10). (B) The immunoprecipitants were analyzed by western blotting. β-actin was used as a loading control. The blots were cropped, and the gels were run under the same experimental conditions. (C) The protein expression levels of B-RAF and RAF1 in the GCs with and without specific siRNA interference were detected by western blotting and normalized for loading by comparison to the signal of β-actin. (D) The granulosa cells were transfected with the reconstructed pYr-adshuttle-4-SLIT2 plasmids and co-infected with or without B-RAF and RAF1 specific siRNA. The negative controls refer to the phosphorylation levels of MEK1/2 and ERK1/2 shown in Fig. 8. −, neither SLIT2 overexpression nor knockdown of RAFs; +, SLIT2 overexpression or knockdown of RAFs. The signal intensity of the phosphorylated proteins was expressed as a ratio to the β-actin signal in arbitrary units (n = 5 per mean ± SEM). The statistical significance is indicated with different superscript characters (P < 0.005).