6 + 4.5%, 83.6 + 4.9% and 65.7 + 4.7%, respectively, in dormant cells. Fig. 5 Peripheral phospho-Y397 FAK localization in dormant cells is integrin α5β1-dependent. a MCF-7 cells were incubated on fibronectin-coated cover slips with medium containing FGF-2 10 ng/ml. Blocking antibodies to integrin α5β1 and integrin α2β1 2 μg/ml and
blocking peptides to fibronectin (P1), to collagen (P3), and a non-binding control RepSox (P2) 100 nM were added on day 3 as described in Materials and mTOR inhibitor Methods. Cells were stained with antibodies to phospho-Y397 FAK on day 6 and photographed at 1,000 x magnification. Localization of phospho-Y397 FAK with dormancy is reversed by blocking fibronectin binding with blocking antibody to integrin α5β1 or blocking peptide P1 to fibronectin. b Graphic depiction of induction of peripheral phospho-Y397 FAK in dormant cells (*p < 0.005),
and reversal of localization by blocking antibody to integrin α5β1 (**p < 0.001) and blocking peptide to fibronectin P1 (***p < 0.01) (Student’s t test). Error bars are + standard deviations. All other GSK458 ic50 differences were not statistically significant. Data is from one of two duplicate experiments with triplicate slides with approximately 100 cells counted per slide To support these data, we immunoprecipitated FAK from lysates prepared from dormant and growing cells and immunostained western blots with antibodies to phospho-Y397 FAK and Protirelin total FAK.
Figure 6a demonstrates that total FAK levels decreased in dormant cells as demonstrated by IP/western blots, while phospho-Y397 FAK levels in the cells slightly increased. The increase in phospho-Y397 was dependent on integrin α5β1, as blocking antibody decreased the rate of this phosphorylation in the IP/westerns, while blocking antibody to integrin α2β1 had no effect. The overall increase in phospho-Y397 FAK was small when whole cellular lysates were assayed by IP/western blot in all of the experiments, while the decreases with integrin α5β1 blocking antibody were consistent. However, the physiologically significant increase in membrane localization of activated FAK was markedly pronounced and significant, as demonstrated by the immunofluorescence staining for phospho-FAK. Fig. 6 Integrin α5β1-dependent peripherally localized phospho-Y397 FAK in dormant cells is associated with membrane localization of the RhoA GAP GRAF. a Cells incubated on fibronectin-coated tissue culture plates with and without FGF-2 10 ng/ml with control or blocking antibodies to integrin α5β1 and integrin α2β1 2 μg/ml added on day 3 were harvested on day 6. Lysates from equal cell numbers were immunoprecipitated with antibody to FAK and stained on western blot with anti-phospho-Y397 FAK antibody, total FAK antibody and GRAF antibody.