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. Author manuscript; available in PMC: 2011 Mar 1.
Published in final edited form as: Oncogene. 2010 Jun 21;29(35):4925–4937. doi: 10.1038/onc.2010.239

Figure 6.

Figure 6

ATF2 suppresses Ha-Ras activation in v-Rel-expressing cells. (a) Activity of the Raf-MEK-ERK pathway in cells expressing v-Rel. Whole cell lysates were prepared from CEFs and DT40 cells expressing control BIS virus (B) and BIS expressing ATF2 (A) and v-Rel alone (V) or in combination (V+A). The expression of phosphorylated ERK (p-ERK), total ERK, phosphorylated MEK1/2 (p-MEK1/2), total MEK1/2, phosphorylated c-Raf (p-c-Raf Ser338), and total c-Raf was determined by Western Blot analysis. The expression of v-Rel and c-Rel in these lysates was demonstrated in Figure 5a. (b) Regulation of Ha-Ras activity by v-Rel and ATF2. Levels of active Ha-Ras in cell lysates from DT40 cells expressing viruses described above were determined using the Active Ras Pull-Down and Detection Kit (Thermo Scientific). The levels of active (top panel) and total (bottom panel) Ha-Ras in these lysates are shown. (c) Regulation of an inhibitory phosphorylation site on c-Raf by v-Rel and ATF2. Western blot analysis was employed to determine the levels of total and phosphorylated c-Raf (Ser259), c-Rel, and v-Rel in whole cell lysates prepared from the CEFs and DT40 cells described above. (d) Reduction in ATF2 levels increases the activation of the Ras signaling pathway. DT40 cells infected with helper virus CSV (H) or viruses expressing v-Rel (V) were superinfected with control RCAS virus (R) or those expressing two different shRNAs against ATF2 (ATF2#1 and ATF2#2). Whole cell lysates were prepared 10 days post-superinfection and analyzed by Western blot for the levels of total and phosphorylated ERK, MEK1/2, and c-Raf as well as ATF2. (e) Dominant negative HA-Ras inhibits ERK activation in v-Rel-expressing cells. CEFs and DT40 cells infected with helper virus CSV (H) or viruses expressing v-Rel (V) were superinfected with control DS virus or viruses expressing a dominant negative Ha-Ras mutant (DN-Ras). Whole cell lysates were prepared and analyzed by Western blot for the levels of total and phosphorylated ERK, DN-Ras, endogenous HA-Ras, c-Rel, and v-Rel. (f) Ha-Ras regulates ERK activation in a v-Rel transformed cell line. 160/2 cells were infected with control DS virus or viruses expressing wild-type Ha-Ras (WT) or DNRas (DN). Whole cell lysates were prepared and analyzed by Western blot for the levels of total and phosphorylated ERK, ectopic Ras (ect. Ras), endogenous Ha-Ras, c-Rel, and v-Rel. (g) Alterations in Ha-Ras activity affect colony formation of v-Rel transformed cells. 160/2 cells described above were plated in soft agar and scored for colony formation after seven days.