RasGRP3 Thr133Ala substitution blocks in vitro phosphorylation by nPKC forms PKCθ and PKCδ. (A) The effect of a RasGRP3 Thr133Ala substitution on phosphorylation by PKCθ is shown. Parental MBP (M), MBP-RasGRP3 (WT), or MBP-RasGRP3 Thr133Ala (T133A) were used as substrates in an immune complex kinase assay, as described in “Materials and methods.” Arrows show, from top to bottom, phosphorylation of MBP-RasGRP3, autophosphorylation of PKCθ, and the position of parental MBP protein. Some lanes are in duplicate. IP indicates immunoprecipitation; Ab, antibody. (B) Similar experiments as in panel A were performed with PKCδ. Results are representative of 3 independent experiments.