p110δ is present in venular endothelium and is activated by TNFα (A) Immunoblots of the p110δ catalytic subunit using recombinant proteins (panel 1) or lysates obtained from HUVECs or neutrophil (panel 2). (B) Representative photomicrograph (3 experiments performed in duplicate) demonstrating phosphorylation of Akt (Ser 243) in response to TNFα-induced activation of HUVECs and inhibition by IC87114. Total Akt present in cells is shown under all conditions tested. (C) Activity of PDK1 in response to TNFα-induced stimulation of vascular endothelium. Lysates from activated HUVECs, pretreated with either vehicle control or the p110δ inhibitor, were subjected to immunoprecipitation using beads coated with an antibody that recognizes PDK1. Enzyme activity was then assessed as described in “Materials and methods.” Relative kinase activity is the average ± SD of 3 experiments with duplicate immunoprecipitates. Activity of PDK1 from untreated cells was represented as 1. P < .01 versus vehicle control. ^ψIC87114 was added to immunoprecipitated PDK1 obtained from TNFα-stimulated HUVEC lysates to assess its ability to directly inhibit this kinase.