H2S Signals through VEGFR-PI3K-Rac1-Cofilin Figure 7. The p110a isoform of PI3K is required for H2S-induced Rac1 activation and endothelial cell migration. (A) HUVECs transfected with p110a, p110b, p110c, p110d or scrambled siRNAs were lysed and analyzed by immunoblotting for PI3K subunits and for Akt phosphorylation on site of Ser473. SiRNA oligonucleotides specifically knocked down expression of their target genes, and GAPDH was used as a loading control. (B) Densitometric analysis of three independent immunoblots to assess Akt phosphorylation levels and the levels of the p110 isoforms following the knockdown experiments. (C) G-LISA assays for Rac1 activation following the action of different p110 isoforms. 50 mM NaHS- induced Rac1 activation was prevented by p110a siRNA. Statistical data (D) and representative micrographs (E) of transwell boyden chamber assays showing that the promotion effect of NaHS on endothelial cell migration was inhibited by p110a siRNA. (G) G-LISA assays for Rac1 activation were also performed following the expression of dominant negative Akt (DN-Akt). Data showing that NaHS (50 mM) induced Rac1 activation cannot be prevented by DN-Akt. (H) The expression of the HA-tag protein demonstrates that cells were successfully transfected. CV, control vector. Data represent the means 6 SE. **P,0.01. *P,0.05. NS, not significant. doi:10.1371/journal.pone.0044590.g007 PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44590