Journal: American journal of human genetics

Article Title: Functional characterization of the 9q34.13 locus identifies RAPGEF1 as a candidate gene modulating risk for melanoma and nevi via RAS activation

doi: 10.1016/j.ajhg.2026.08.014

Figure Lengend Snippet: C283T immortalized melanocytes with doxycycline-inducible expression of RAPGEF1 were grown in supplement-free medium for 24hrs and subsequently treated with human EGF (hEGF) protein for the indicated time. A . Active GTP-bound RAP1 was pulled down by RalGDS-RBD beads from whole-cell extract. Pull-down and whole-cell (total RAP) fractions were compared by Western blotting with an antibody against RAP1. B . Active GTP-bound RAS was pulled down by Raf-RBD beads, with the pull-down and whole-cell (total RAS) fractions compared by Western blotting with an antibody against RAS. Active and total AKT and ERK were compared via Western blotting. The intensity of the phospho- versus total-AKT and ERK was determined by the ratio of the band intensity. Figures are representative experiments from three biological replicates.

Article Snippet: RAP1 activation was measured using the RAP1 activation assay kit from Abcam (ab212011) and RAS activation was measured using GST-fusion Raf-1 RBD beads (Sigma, #14863) or RAF-RBD Protein GST Beads (RF02-A, Cytoskeleton).

Techniques: Activation Assay, Expressing, Pull Down Assay, Western Blot