RET/PTC-mediated ERK activation is dependent on RAS and BRAF. (A) Schematic representation of the RET, HRAS, and BRAF constructs. The RET/PTC breakpoint and RET tyrosines 1015 and 1062 are indicated. Residues V600 and K483 are mutated to E and M, respectively, in the BRAF(V600E) and BRAF(K^–) plasmids. Residues G12 and S17 are mutated to V and N in the HRAS(V12) and HRAS(N17) plasmids. C, CAAX tail; CR1–3, conserved BRAF regions 1–3; Cys, cysteine-rich; EC, extracellular domain; ED, HRAS effector domain; H, heterogeneous region; JX, juxtamembrane; SP, RET signal peptide; TK, tyrosine kinase; TM, transmembrane. (B) Protein lysates (500 μg) extracted from HEK293 cells transfected with the indicated plasmids underwent immunoprecipitation with anti-tag (myc) antibody. Kinase assay was performed with GST-MEK as a substrate. BRAF and RET/PTC3 were detected by Western blotting (W.B.) with anti-myc and anti-RET antibodies, respectively. RAS expression was detected with an anti-RAS monoclonal antibody that also recognizes the endogenous protein. (C) HEK293 cells transfected with the indicated plasmids were harvested, and protein extracts were subjected to immunoblotting with anti–phospho-p44/p42 ERK (pERK) antibodies. The blot was reprobed with anti-p44/p42 antibodies for normalization. RET/PTC3 and RAS were detected by Western blotting with specific antibodies. These experiments are representative of at least 3 independent assays. (D) Transient BRAF suppression was achieved by RNA interference. Whole cell lysates were prepared 48 hours after transfection and analyzed for protein expression by Western blotting with the indicated antibodies. siRNA(SCR), scrambled siRNA.