Fig. 3: TFG-RET exhibits kinase activity and forms oligomeric complexes.

a HeLa cells were transiently transfected with V5-tagged wild-type RET, TFG-RET and kinase dead TFG-RET K270M along with empty vector. After 48 h cells were lysed and subjected to immunoprecipitation using anti-V5 antibody. After subsequent washes, the immunoprecipitated proteins were used for in vitro kinase assay, as described in Methods. TFG-RET phosphorylated MBP, thus exhibiting kinase activity like wild-type RET kinase. This kinase activity was abrogated in the TFG-RET kinase dead mutant (p, phosphorylated). Shown are representative data from at least three independent experiments. b HeLa cells were co-transfected with FLAG-tagged and V5-tagged wild-type RET and TFG-RET as indicated, followed by anti-FLAG immunoprecipitation after 48 h. Lysates were analyzed by western blotting (TCL, total cell lysate). Wild-type RET (FLAG®) co-immunoprecipitated with wild-type RET (V5) and also with TFG-RET (V5) (left panel), similarly, TFG-RET (FLAG®) also co-immunoprecipitated with both wild-type RET (V5) and TFG-RET (V5). Empty vector controls were included. Shown are representative data from at least three independent experiments. c Nthy-ori 3-1 cells stably expressing wild-type RET and TFG-RET were treated with DTME and DTME + DTT. Upon chemical crosslinking with DTME, similar to wild-type RET, TFG-RET also formed high molecular weight heteromeric complexes and these complexes were disrupted upon reduction with DTT (lane 3 and 6). Shown are representative data from at least two independent experiments.