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. 2014 Oct 6;111(42):15155–15160. doi: 10.1073/pnas.1417549111

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Fig. 5. — Activation of the Raf/Mek/Erk pathway is essential for cell proliferation in the absence of the p53, p21Cip1, or Rb tumor suppressor. (A) Western blot analysis of phospho (p)-Erk1/2, Erk1/2, p-Mek1/2, Mek2, p-Elk-1, Elk-1, and K-Ras expression in K-Raslox MEFs left untreated (−4OHT) and in Rasless MEFs generated by treatment with 4OHT for 2 wk (+4OHT) expressing shp53-A (shp53), shp21-B (shp21), or the SV40T121 oncoprotein (T121). GAPDH expression served as a loading control. (B) qRT-PCR analysis of the relative expression levels of the indicated mRNAs in K-Raslox MEFs left untreated (−4OHT) and in Rasless MEFs generated by treatment with 4OHT for 2 wk (+4OHT) expressing a p53-specific shRNA (shp53-A), a p21Cip1-specific shRNA (shp21-B), or the SV40T121 oncoprotein (T121). β-Actin expression levels were used for normalization. Data are represented as mean ± SD. **P < 0.01; ***P < 0.001 (unpaired Student t test). (C) Colony formation of Erkless (Erk1^−/−;Erk2^−/−) and Erklox (Erk1^−/−;Erk2^lox/lox) MEFs expressing the indicated cDNAs or shRNAs and represented as the ratio between the number of colonies observed in cells lacking the Erk1/2 proteins (Erkless) and those cells expressing Erk2 (Erklox). Data are represented as mean ± SD. (D) Colony formation of Mekless (Mek1^−/−;Mek2^−/−) and Meklox (Mek1^lox/lox;Mek2^−/−) MEFs expressing the indicated cDNAs or shRNAs and represented as the ratio between the number of colonies observed in cells lacking the Mek1/2 proteins (Mekless) and those cells expressing Mek1 (Meklox). Data are represented as mean ± SD. (E) Colony formation of Rafless (A-Raf^−/−;B-Raf^−/−;c-Raf^−/−) and Raflox (A-Raf^lox/lox;B-Raf^lox/lox;c-Raf^lox/lox) MEFs expressing the indicated cDNAs or shRNAs and represented as the ratio between the number of colonies observed in cells lacking the A-Raf, B-Raf, and c-Raf proteins (Rafless) and those cells expressing the three Raf proteins (Raflox). Data are represented as mean ± SD.