FIGURE 3.

R454 FAK expression and FAK phosphorylation target identification within E8.5 embryo lysates and establishment of primary FAK^R454/R454 MEFs. A and C, protein lysates from FAK^WT/WT (WT) and FAK^R454/R454 (R454) embryos harvested at E8.5 or lysates from primary MEFs were analyzed by immunoblotting for total FAK, Tyr(P)-397 (pY397) FAK, total Pyk2, Pyk2 Tyr(P)-402 (pY402), total c-Src, and Src Tyr(P)-416 (pY416). Anti-phosphotyrosine (pY) immunoblotting revealed decreased target protein tyrosine phosphorylation (asterisks) in lysates from E8.5 R454 embryos. Anti-actin immunoblotting was used as a loading control. Decreased c-Src activation (as detected by Src Tyr(P)-416 blotting) was detected in R454 embryos and MEFs. A, p53 tumor suppressor expression was lower in R454 E8.5 embryos as determined by immunoblotting. B, phase-contrast images of E8.5 FAK^R454/R454 embryo outgrowth in culture after 2 and 7 days. Shown is Matrigel-embedded embryo (dark center) and surrounding cell outgrowth. Bar, 100 μm. D, primary WT and R454 MEFs proliferate equally in culture. Results are the mean cell number (n = 3 independent points ± S.D. (error bars)).