Generation of thyroid cell cultures. (A) Protein lysates (50 μg) extracted from the indicated cell lines underwent Western blotting with anti-RET, -tag (myc), or -RAS antibodies. Equal protein loading was ascertained by anti-tubulin immunoblot. (B) Mass populations of PC cells transfected with the indicated plasmids were photographed using a phase-contrast light microscope (magnification, ×150). RET/PTC3-, RET/PTC3(Y1015F)-, BRAF(V600E)-, and HRAS(V12)-expressing cells displayed a transformed morphology, whereas RET/PTC3(Y1062F)-expressing cells were virtually indistinguishable from parental cells. (C) Protein extracts from the indicated cell lines were subjected to immunoblotting with anti–phospho-p44/42 ERK antibodies. The blot was reprobed with anti-p44/42 antibodies for normalization. (D) BRAF targeting by siRNA but not by scrambled siRNA induced BRAF downregulation and ERK inhibition, as shown by Western blotting with specific antibodies.