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Fig. 3 | BMC Cancer

Fig. 3

From: An oncogenic mutant of RHEB, RHEB Y35N, exhibits an altered interaction with BRAF resulting in cancer transformation

Fig. 3

Cell Lines Stably Expressing RHEB Y35N Activate RAF/MEK/ERK Signaling Similar to KRAS G12 V Cell Lines. (a) NIH 3 T3 cell lines stably expressing RHEB WT, RHEB Y35N, or KRAS G12V were generated using lentiviral transduction. Left: Western blot of FLAG, RHEB, KRAS, and ACTIN protein in total cell lysates collected from NIH3T3 cell lines stably expressing RHEB WT, RHEB Y35N, KRAS G12V, or an empty vector expressing no protein (Ctrl). Right: Fold expression for RHEB was calculated by identifying all band intensities using ImageJ analysis, then finding the ratio of RHEB/ACTIN for each sample. The Ctrl sample RHEB/ACTIN ratio was set at 1. All other samples’ ratios were normalized to Ctrl. KRAS fold expression was calculated same as RHEB. Graph represents the averages of experiments (n = 2). b NIH 3T3 cell lines stably expressing RHEB WT, RHEB Y35N, KRAS G12 V, or Ctrl were grown in the presence or absence of serum, and cell lysates were collected after 24 h. Western blot was performed using antibodies for phosphorylated –ERK, and total –ERK. Fold expression for pERK was calculated as in (a). Briefly, band intensities as determined by ImageJ were used to find the ratio of pERK/total ERK for each cell line and condition. The ratio for Ctrl under normal growth conditions was set to 1, and all other ratios were normalized to Ctrl under normal growth conditions. Graph represents the averages of experiments (n = 2). c Same experiment as performed in (b), except looking at phosphorylated –S6, and total –S6. Fold expression for pS6 was calculated as in (b). d NIH 3 T3 cell lines stably expressing Y35N were transiently transfected with plasmids to overexpress RHEB WT. Left: Western blot for phosphorylated-ERK (pERK) is shown. Right: Graph showing ERK activation based on levels of phosphorylated ERK compared to total ERK. The pERK/totalERK ratio for RHEB Y35N and RHEB Y35N + RHEB WT cell lines, was determined by quantifying the intensities of the Western blot bands for pERK and totalERK using imageJ. The graph shows results from three separate experiments

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