Figure 8.

UV does not promote activation and dimerization of extracellular domain–deleted Ret whose cysteine 376 was replaced by alanine. Lysates from NIH 3T3 cells transfected with original RET-PTC-1 (D and E, lanes 4), RET-PTC-1–C365A (A and C, lane 1; D, lanes 1–3), and RET-PTC-1–C376A (B and C, lane 2; E, lanes 1–3) after sham or UV irradiation were analyzed by either in vitro kinase assay after immunoprecipitation with anti-Ret antibody (A and B) or Western blotting with anti-Ret antibody (C–E). SDS-PAGE was done under reducing (A–C) or unreducing (D and E) conditions in 8% (C–E) or 13% (A and B) polyacrylamide gels. (A and B) Lanes 1, sham irradiation; lanes 2–4, 5 min (lane 2), 10 min (lane 3), and 15 min (lane 4) after UV-B irradiation (600 J/m²). (D and E) Lanes 1 and 4, sham irradiation; lanes 2 and 3, 5 min (lane 2) and 15 min (lane 3) after UV-B irradiation (600 J/m²). pRet, autophosphorylated Ret-PTC-1–C365A (A) or Ret-PTC-1–C376A (B); pMBP, phosphorylated MBP; M, monomer Ret; D, dimer Ret. All measurements were repeated three times with basically the same results. Representative results are provided.