Figure 5.

UV promotes dimerization of c-Ret. Lysates from NIH 3T3 cells transfected with c-RET after sham or UV irradiation were subjected to Western blotting with anti-Ret antibody (A and B) or to in vitro kinase assay (C–E) after immunoprecipitation with anti-Ret antibody. SDS-PAGE was done under reducing (A, C, and E) or unreducing (B and D) conditions in 5% (A–D) or 13% (E) polyacrylamide gels. (A–D) Lanes 1, sham irradiation; lanes 2, 5 min after UV-B irradiation (600 J/m²). (E) Ret proteins were immunoprecipitated from the lysate of the transfectants that had received UV-B irradiation (600 J/m²) 5 min earlier. The immunoprecipitated Ret proteins were untreated (lane 1) or treated with 5% 2ME for 30 min (lane 2), washed five times to remove 2ME, and then subjected to in vitro kinase assay. pRet (doublet band), autophosphorylated c-Ret; pMBP, phosphorylated MBP; M, monomer Ret; D, dimer Ret. All measurements were repeated three times with basically the same results. Representative results are provided.