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. 2014 Nov 15;6(1):221–233. doi: 10.18632/oncotarget.2771

Figure 3. Silencing of truncated murine Raf1 restores sensitivity to PHA.

Figure 3

(A) Western Blot analysis on resistant Mouse Testis (MmT) GTL-16 cells, transduced with non silencing scramble vector (scr) or two different murine-specific shRNAs against Raf1: shRNA-5′, targeting the 5′ portion of the Raf1 transcript, and shRNA-3′, targeting the 3′ portion. The protein was detected by an antibody against the C-terminus of Raf1 (bands with lower molecular weight respect to the ~60kD truncated RAF1 band are non-specific and appear also in non-transduced cells); vinculin was used as loading control. (B) Realtime PCR validation of murine Raf1 mRNA specific silencing by the shRNA-3′ construct. The y-axis represents the percentage of inhibition in scramble and shRNA-3′ compared to resistant MmT GTL-16 cells (SEL); Pgk1 housekeeper gene was used as control. (C) Resistant GTL-16 MmT cells, either untransduced (MmT SEL) or transduced with non-silencing shRNA (scramble) or shRNA-5′ or shRNA-3′ silencing constructs were treated with increasing concentrations of PHA (as indicated) for 1 weeks and subsequently fixed, stained, and photographed.