FIG. 1.
Significant accumulation of tyrosine phosphates on kinase-inactive Raf-1. (A) Sf21 insect cells were infected with recombinant baculoviruses encoding either the wild-type Raf-1 (Raf-1) or kinase-inactive Raf-1 (301), alone or in combination with baculoviruses encoding Jak2 or pp60vsrc (Src). Anti-Raf-1 immunoprecipitates were analyzed by SDS-PAGE (8% gel), immunoblotted with antiphosphotyrosine (anti-P-tyr in all figures) antibody, and then reprobed with anti-Raf-1 antibody. The blots were developed by enhanced chemiluminescence. (B) NIH 3T3 cells were transfected with wild-type Raf-1 or with kinase-inactive mutant 301; 48 h posttransfection, cells were either unstimulated (−) or stimulated (+) with PDGF for 10 min. The expressed Raf-1 was immunoprecipitated and Western blotted with antiphosphotyrosine antibody 4G10 (top). Raf-1 expression was detected by probing the identical membrane with anti-Raf-1 antibody (bottom).