How CD151 maintains the balance of RhoA and Rac1. (A-B) CD151 does not affect p190Rho^GAP and p115Rho^GEF activation. (A) Cellular p190Rho^GAP was immunoprecipitated with p190Rho^GAP mAb. The amount of tyrosine-phosphorylated p190Rho^GAP was determined by immunoblotting with the phosphotyrosine mAb PY99. After stripping, total precipitated p190Rho^GAP was detected by p190Rho^GAP mAb. The active form of p190Rho^GAP (A) or p115Rho^GEF (B) was detected by a GST pulldown assay using a GST-tagged, constitutively active RhoA mutant (Q63L) or GST-tagged, nucleotide-empty RhoA mutant (G17A), respectively. (C) CD151 silencing reduces the cAMP level in ECs. HMEC transductants were lysed in 0.1N HCl. cAMP contents in lysates were measured using a cAMP EIA kit. *P < .05. (D) 8-Br-cAMP, but not 8-CPT-cAMP, stabilizes the network structures of CD151-silenced ECs. HMEC transductant cells were incubated with 8-Br-cAMP (500μM) or 8-CPT-cAMP (500μM) for the endothelial network formation assay. The images were taken 18 hours after incubation. Bar represents 250 μm. (E) CD151 silencing reduced PKA-phosphorylated GSK-3β in ECs. The phosphorylated GSK-3β at Ser9 residue in HMEC lysates was detected by Western blot using an anti–phospho-GSK-3β (Ser9) Ab and quantified by densitometry (mean ± SE, n = 4). *P < .05. The treatment of PI3K inhibitor LY294002 did not alter the difference in GSK-3β (Ser9) phosphorylation between MOCK and CD151 KD groups.