Figure 3.

A: Schematic representation of RET constructs used in this study. SP, signal peptide; EC, extracellular domain; CYS, cysteine-rich region; TM, transmembrane region; JM, juxtamembrane domain; TK, tyrosine-kinase domain. Autophosphorylated RET tyrosines 905, 1015, and 1062 are shown. B: The hormone-independent proliferation of the various PC Cl 3 transfectants and empty-vector transfected cells (−) is reported as percentage of G418-resistant cells forming colonies after 15 days of incubation in the absence of 6H. Average results of three independent experiments ± SD. C: Expression of the various RET proteins in transfected or empty vector-transfected (−) PC Cl 3 cell clones. Fifty μg of protein lysates were analyzed by Western blot with anti-RET(TK) antibodies. Full-length RET proteins formed a doublet of 170 kd and 150 kd of relative molecular mass. Antibodies directed against tubulin were used for normalization. D: Parental and transfected PC Cl 3 cells were photographed by using a phase-contrast light microscope. E: The anchorage-independent growth of the various PC Cl 3 transfectants and parental cells was evaluated in comparison to the ARO cell line, used as a positive control. F: The expression of thyroid differentiated genes in parental and transfected PC Cl 3 cells was evaluated by semiquantitative RT-PCR. Reverse-transcribed cDNAs were PCR amplified with primers for TG and PAX-8. β-Actin levels are shown to confirm equal cDNA content. Original magnification, ×150 (D).