Fig. 4.

Upregulated RNF44 expression is related to hyperactivation of ERK and AKT in BR cells. (A) Immunoblotting demonstrated that BR and BMR cells possessed downregulated AMPK-α1 and upregulated RNF44. (B) RNA levels of RNF44 in parental and BR cell lines were determined by qRT-PCR, and normalized by GAPDH. (C) Higher protein levels of p-AKT, p-ERK, and RNF44 appeared in BR cells even in presence of BRAFi (vemurafenib). (D) BR cell lines were treated with MEKi (trametinib, 5 nM), ERKi (SCH772984, 3 μM), or AKTi (MK-2206, 2 μM) for 24 hr, and their RNF44 levels were determined by qRT-PCR. Data shown in a bar graph were represented as mean ± SEM (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.005). (E) Protein levels of ERK, AKT, AMPK-α1, and RNF44 in BR and BMR cells were determined by immunoblotting following treatment with AKTi or ERKi.