Figure 2

Physical association between PLD and RET/PTC1. (A) HEK293 cells were transfected with flag-tagged-PLD1 or -PLD2 and RET/PTC1 expression plasmids. After 24 h transfection, whole cell lysates were immunoprecipitated with anti-RET (upper panel) or anti-flag (lower panel) antibodies, and then immunoprecipitants were analyzed by Western blotting with anti-flag or anti-RET antibodies. The efficiency of immune-precipitant was measured with anti-RET and anti-flag antibodies (each bottom panel). (B) TPC-1 cells were transfected with RET/PTC1 expression plasmid, and whole cell lysate was immnunoprecipitated with antibodies directed against RET and normal IgG. Immunoprecipitates were analyzed by Western blotting with anti-PLD antibody (upper panel). The efficiency of immune-precipitant was measured with anti-RET antibody (bottom panel). One percent of cell extracts from sample was used as a control of protein input. (C) HEK293 cells were transfected with various combinations of plasmids encoding an empty vector, RET/PTC1 plus either flag-tagged wild-type (wt)- or lipase inactive mutant (KRM)- PLD2. Cell lysates were immunoprecipitated with anti-flag antibody, and then immunoprecipitants were analyzed by Western blotting with anti-RET antibody (upper panel). The efficiency of immune-precipitant was measured with anti-flag antibody (bottom panel).