Figure 3. Constitutive activation of FAK in cells expressing the FP-E132T receptor.

Cells expressing wild-type FP-R or the indicated receptor mutant were incubated for 5 min in the presence or absence of 1 μM PGF_2α, washed with PBS buffer and then lysed. The lysate was centrifuged and the supernatant was subjected to an immunoprecipitation with an FAK-specific antibody. Immunoprecipitated proteins were separated by SDS/PAGE and blotted on to PVDF membranes. Proteins were quantified with a phosphotyrosine-specific antibody (pFAK) and after stripping and restaining with an FAK-specific antibody, an appropriate peroxidase-labelled secondary antibody and a chemiluminescence substrate. Total (FAK) and phosphorylated (pFAK) immunoreactivities on the same blot are given. Values were normalized to the values of FAK and pFAK in the absence of PGF_2α in cells expressing the wild-type (Wt) receptor. They are means±S.E.M. for three independent experiments. Statistics: Student's t test; a, significantly different from wild-type non-stimulated control; and b, significantly different from respective non-stimulated cells.