Pak2 and Pak4 act downstream of SFKs to coactivate Raf kinases that are
involved in EC lumen formation in 3D collagen matrices. (A) ECs were
resuspended in 3D collagen matrices in the absence or presence of PP2 (10
μM). Extracts were prepared at 24 hours for western blot analysis and
probed for phospho-Pak2, phospho-Pak4 or actin. (B) ECs containing GFP- or
Csk-expressing adenoviruses (Ad) were resuspended in 3D collagen matrices.
Extracts were made at 24 hours for western blot analysis and probed for
phospho-Pak2, phospho-Pak4 or actin. (C) ECs treated with the indicated siRNAs
were resuspended in 3D collagen matrices. Extracts were made at 24 hours for
western blot analysis and probed for phospho-Pak2, phospho-Pak4 or actin. (D)
ECs treated with the indicated siRNAs or adenoviruses [GFP, DN Pak2 (T402A) or
DN Pak4 (K350M)] were resuspended in 3D collagen matrices. Extracts were made
at 24 hours for western blot analysis and probed for phospho-B-Raf,
phospho-C-Raf or actin. (E) Extracts of EC cultures in 3D collagen matrices
were prepared at the indicated time points and probed for phospho-B-Raf or
phospho-C-Raf. Actin, B-Raf and C-Raf were used as loading controls.