Ang-1 activates PKC independently of PLCγ, but does not inhibit VEGFR-2 activation and signaling. (A) BAEC were stimulated with VEGF (40 ng/mL), Ang-1 (100 ng/mL), or both simultaneously for the indicated times. VEGFR-2 and Tie2 tyrosine phosphorylation levels were monitored in VEGFR-2 and Tie2 immunoprecipitates (IP). Equal immunoprecipitation levels from BAEC lysates were confirmed by Western blot (wb). supplemental Figure 2 shows the complementary treatments and IPs. These experiments were repeated at least 3 times with identical results. (B) Activation of Akt, MAPK, PKC, and PLCγ, using the indicated phospho-specific antibodies, was monitored in BAEC stimulated with Ang-1 (100 ng/mL), VEGF (40 ng/mL), or both simultaneously for the indicated times. Equal protein loading was confirmed by reprobing the membranes with antibodies against total Akt, MAPK, and PLCγ. These experiments were repeated at least 3 times with similar results.