PKA inhibits angiogenesis by inducing apoptosis. (a) CAMs stimulated with bFGF were transfected 24 hours later by placing 4 μg pcDNA/V5/His dnPKA or N1-GFP expression plasmid on CAMs. CAMs were treated on the same day with saline or anti-α5β1 Ab’s and were harvested 48 hours later. Blood vessel branch points were quantified. (b) Cryosections of CAMs from a were immunostained with anti-vWF (red) and were stained to detect fragmented DNA by the TUNEL method (green). Arrows indicate blood vessels. Apoptotic blood vessels appear yellow. (c) Western blots of lysates prepared from CAMs treated as in a were immunoblotted with anti–cleaved caspase-3 and anti–cleaved caspase –8, as well as anti-actin as a loading control. (d) CAMs stimulated with bFGF were treated with saline or 250 μM cAMP or were transfected by placing 4 μg pcDNA/V5/His PKA_cat or N1-GFP expression plasmid on stimulated CAMs. Blood vessel branch points were quantified 48 hours later. (e) Cryosections of CAMs from d were immunostained with anti-vWF (red) and were stained to detect fragmented DNA by the TUNEL method (green). Arrows indicate blood vessels. Apoptotic blood vessels appear yellow. (f) Detergent lysates prepared from freshly excised CAMs from d were immunoblotted for expression of cleaved caspase-3 and actin as a loading control. (g) Cryosections of CAMs from a and d were immunostained with anti-pentaHis (red) to detect expression of His-tagged transgenes in the transfected CAM tissue. Sections were counterstained with DAPI. Arrows indicate blood vessels.