FIG. 4.

Investigation of the targets of phosphorylation. (A) Phosphate was incorporated into COS-7 PLD2 by the action of EGFR, Src, or JAK3 kinases in vitro. PLD2 α-myc-agarose-bound immunocomplexes were used for measurement of PO₄ incorporation into PLD2. The results represent means ± the SEM of three independent experiments in duplicate. *, differences between means that were statistically significant (P < 0.05) as determined by ANOVA. (B) SDS-gel/autoradiograph of EGFR phosphorylated PLD2. Following kinase activations with or without [³²P]ATP, proteins were electrophoresed and transferred onto PVDF membranes. Radioactive, phosphorylated PLD2 was detected by autoradiography. (C) Western blotting of EGFR phosphorylated PLD2. Nonradioactive, phosphorylated PLD2 was detected by using α-myc rabbit antibodies. (D) Representative autoradiograph of Src (first panel) or JAK3 (second panel). A representative Western blot of mycPLD2 (third panel) is also shown to indicate equal loading of the gels.