Fig. 2.

Akt mediates R-Ras-dependent microtubule stabilization and lumenogenesis. a R-Ras activates Akt isoforms. Ser473 phosphorylation of Akt1 and Akt2 were determined by western blot using phospho-specific antibody for each isoform as well as by immunoprecipitation of Ser473-phosphorylated Akt followed by western blot for each Akt isoform. b The effects of R-Ras on Akt Ser473 and GSK-3β Ser9 phosphorylation and α-tubulin acetylation were examined by western blot. c PI3K inhibition by LY294002 blocks the effects of R-Ras38V. d Endothelial sprouts in 3-D cultures of R-Ras38V-transduced, Rictor, or Raptor-silenced ECs. ECs were transduced with R-Ras38V followed by Rictor or Raptor knockdown using siRNA (siRictor or siRaptor) before embedded in fibrin gel. Arrows, R-Ras-induced lumenogenesis was unaffected by Raptor knockdown. e, f Constitutive activation of GSK-3β blocks R-Ras-dependent microtubule stabilization and lumenogenesis. ECs were first transduced with/without R-Ras38V and subsequently transduced with/without GSK-3β S9A. Acetylation of α-tubulin was analyzed by western blot (e) and immunofluorescence (f) and lumenogenesis examined in 3-D culture (f). g–i Akt1 and Akt2 are essential to the R-Ras-mediated GSK-3β inhibition, microtubule stabilization, and lumenogenesis in vitro. Either Akt isoform was silenced in R-Ras38V-transduced ECs by siRNA (siAkt1 or siAkt2). GSK-3β phosphorylation and α-tubulin acetylation were examined by western blot (g) or western blot and immunofluorescence (g, h). The effect of Akt silencing on R-Ras-mediated EC lumenogenesis was examined in 3-D cultures (i). Arrowheads, severely disrupted lumen formation. Scale bars, 50 µm (d, f 3-D culture, h, i), 25 µm (f2-D culture)