Skip to main content
. Author manuscript; available in PMC: 2016 May 3.
Published in final edited form as: Cancer Cell. 2011 Apr 21;19(5):652–663. doi: 10.1016/j.ccr.2011.04.002

Figure 8. B-Raf and c-Raf are not essential for proliferation and immortalization of primary MEFs driven by wild type or oncogenic K-Ras signaling.

Figure 8

(A) Western blot analysis of B-Raf and c-Raf protein expression in primary MEFs derived from (top) three independent K-Ras+/G12V;RERTert/ert, K-Ras+/G12V;B-Raflox/lox,RERTert/ert, K-Ras+/G12V;c-Raflox/lox;RERTert/ert and K-Ras+/G12V;B-Raflox/lox;c-Raflox/lox;RERTert/ert embryos and (bottom) three independent K-Ras+/+;RERTert/ert, K-Ras+/+;B-Raflox/lox;RERTert/ert, K-Ras+/+;c-Raflox/lox;RERTert/ert and K-Ras+/+;B-Raflox/lox;c-Raflox/lox;RERTert/ert embryos. These MEFs were incubated in DMEM media supplemented with 10% FBS in the presence of 4OHT for 5 days to activate expression of the resident K-RasG12V oncogene and to eliminate the c-Raflox and/or B-Raflox conditional alleles. Gapdh served as loading control.

Migration of the corresponding proteins is indicated by arrowheads.

(B) Growth curves of the primary MEFs (n=3) described above. MEFs were grown in DMEM media supplemented with 10% FBS and 600 nM 4OHT during 5 days before seeding. Results are shown in arbitrary absorbance units.

Left panel: K-Ras+/G12V;RERTert/ert (solid circles), K-Ras+/G12V;B-RafΔ/Δ;RERTert/ert (open squares), K-Ras+/G12V;c-RafΔ/Δ;RERTert/ert (open circles), K-Ras+/G12V;B-RafΔ/Δ;c-RafΔ/Δ;RERTert/ert (open triangles).

Right panel: K-Ras+/+;RERTert/ert (solid circles), K-Ras+/+;B-RafΔ/Δ;RERTert/ert (open squares), K-Ras+/+;c-RafΔ/Δ;RERTert/ert (open circles), K-Ras+/+;B-RafΔ/Δ;c-RafΔ/Δ;RERTert/ert (open triangles).

Error bars indicate +/− SD of the mean.

(C) (Left) Western blot analysis of B-Raf and c-Raf protein expression in primary MEFs derived from five independent K-Ras+/G12V;B-Raflox/lox;RERTert/ert, K-Ras+/G12V;c-Raflox/lox;RERTert/ert and K-Ras+/G12V;B-Raflox/lox;c-Raflox/lox;RERTert/ert embryos. Samples from two K-Ras+/G12V;RERTert/ert embryos are also shown. MEFs were incubated in DMEM media supplemented with 2% FBS in the presence of 4OHT for 5 days to activate expression of the resident K-RasG12V oncogene and to eliminate the c-Raflox and/or B-Raflox conditional alleles. Gapdh served as loading control. Migration of the corresponding proteins is indicated by arrowheads.

(Right) Growth curves of the primary MEFs (n=5) described above. MEFs were grown in DMEM media supplemented with 2% FBS and 600 nM 4OHT. K-Ras+/G12V;RERTert/ert (solid circles), K-Ras+/G12V;B-RafΔ/Δ;RERTert/ert (open squares), K-Ras+/G12V;c-RafΔ/Δ;RERTert/ert (open circles), K-Ras+/G12V;B-RafΔ/Δ;c-RafΔ/Δ;RERTert/ert(open triangles).

Error bars indicate +/− SD of the mean.

(D) Immortalization of the primary MEFs described in (B) following a 3T3 protocol (n=3). Cells were cultivated in DMEM media supplemented with 10% FBS and 600 nM 4OHT. Symbols are those described in (B).

Error bars indicate +/− SD of the mean.

See also figure S8.