Figure 2.

Functional Assays of RRAS2 Variants

(A) Representative immunoblots of three independent experiments. HEK293 cells transfected with WT or mutant RRAS2 constructs or empty vector (Mock) were used for pull-down assays and immunoblotting. RRAS2-guanosine triphosphate (GTP) which was pulled down using RAF1-RBD agarose, total RRAS2, phospho-MEK1/2, total MEK1/2, phospho-ERK1/2, total ERK1/2, and β-actin as a loading control were shown. WT, wild-type.

(B) Stimulation of ELK transcription by RRAS2 mutants. ELK-GAL4 and GAL4-luciferase trans-reporter vectors were transiently co-transfected with RRAS2 constructs into unstimulated HEK293 cells. Relative luciferase activity (RLA) was normalized to the activity of a co-transfected control vector (phRLnull-luc) expressing Renilla reniformis luciferase. Folds under each bar were calculated as a multiple of WT. Data are presented as mean ± SD; n = 3 per group. WT, wild-type. ^∗∗∗p < 0.001 compared with WT.