Figure 9.

Overexpression of SOD1 in cells prevents UV-mediated superactivation of Ret. Lysates from the HA-SOD1− (A and B, lanes 1, and C) or HA-SOD1+ (A and B, lanes 2, and D and E) NIH 3T3 cells transfected with Ret-MEN2A were analyzed by either Western blotting with anti-HA (A) or anti-Ret antibody (B and E) or in vitro kinase assay (C and D) after immunoprecipitation with anti-Ret antibody. SDS-PAGE was done under reducing (A–D) or unreducing (E) conditions in 5% (E), 10% (A and B), or 13% (C and D) polyacrylamide gels. (C–E) 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). pRet (doublet band), autophosphorylated Ret-MEN2A with (C) or without (D) SOD1; pMBP, phosphorylated MBP; M, monomer Ret; D, dimer Ret. All measurements were repeated three times with basically the same results. Representative results are provided.