Figure 3.

UV irradiation induces superactivation of mutant Ret. Lysates from the NIH 3T3 cells transfected with c-RET (A–C), RET-MEN2A (D–F), or RET-MEN2B (G–I) after sham or UV irradiation were immunoprecipitated with anti-Ret antibody, and the resulting immunoprecipitates were subjected to either Western blotting with anti-phosphotyrosine antibody (A, D, and G) or anti-Ret antibody (B, E, and H) or in vitro kinase assay (C, F, and I). SDS-PAGE was done in 5% (A, B, D, E, G, and H) or 13% (C, F, and I) polyacrylamide gels. Lanes 1, sham irradiation; lanes 2, 5 min after UV-B irradiation (600 J/m²). pRet (doublet band), autophosphorylated c-Ret (C), Ret-MEN2A (F), or Ret-MEN2B (I); pMBP, phosphorylated MBP. All measurements were repeated three to four times with basically the same results. Representative results are provided. The exposure times to x-ray film for autoradiography for A, D, and G and for C, F, and I were adjusted to develop bands with comparable density for lanes 1 as sham-irradiated control; see Figure 2 for comparison of the density of bands among c-Ret, Ret-MEN2A, and Ret-MEN2B that were developed by the same exposure time.