FAK and AKT phosphorylation. Serum-starved wild-type MLECs were plated on FN-coated dishes in incomplete medium (ICM) supplemented with and without α3(IV)NC1 or α3β1+α3(IV)NC1 or αVβ3+α3(IV)NC1, or α3β1+αVβ3+α3(IV)NC1, for 0 to 60 minutes and cytosolic extracts were analyzed by Western immunoblotting. (A) Immunoblots of phosphorylated FAK (p-FAK, upper blot) and total signaling FAK protein (FAK, lower blot). (B) Phosphorylated AKT (p-AKT, upper blot) and total signaling AKT protein (AKT lower blot). (C-E) Similar to panels A and B but with and without α3(IV)NC1 or α3β1+α3(IV)NC1, or αVβ3+α3(IV)NC1, or α3β1+αVβ3+α3(IV)NC1. (F-H) Regulation of IκB-α and NFκB in ECs by α3(IV)NC1. (F) Hypoxic cell extracts were immunoblotted with phosphorylated IκB-α (p-IκB-α, upper blot) and total IκB-α protein (lower blot). (G) Nuclear translocations of NFκB staining in MLECs were determined using a Zeiss LSM 510 Meta Confocal microscope (Carl Zeiss) with a Plan-Neo 63×/1.4 NA objective lens. Images were captured using a Flex Digital IEEE-1394 camera (Sheerin Scientific), and processed using SPOT advanced imaging software version 3.1.0 (Diagnostic Instruments) and Adobe Photoshop 7.0 (Adobe). (H) Immunoblots of cytosolic and nuclear extracts showing the NFκB translocation.