Figure 4

Focal adhesion kinase (FAK) activity coordinates the formation of β₃ integrin:TβR-II complexes. (a) β₃ integrin was immunoprecipitated (β₃ I.P.) from control (i.e., GFP) or β₃ integrin-expressing NMuMG cell extracts, and the resulting immunocomplexes were immunoblotted with antibodies against TβR-II and FAK, as indicated. Direct immunoblot analysis of an aliquot of the prepared cell extracts (Input) served to monitor the total levels of TβR-II, β₃ integrin, and β-actin. Data are representative images from a single experiment that was repeated 3 times and show that FAK was present in β₃ integrin:TβR-II complexes. (b) β₃ integrin (β₃ I.P.) or TβR-II (TβR-II I.P.) were immunoprecipitated from control (i.e., scram) and FAK-deficient (shFAK) NMuMG cell extracts before (pre-EMT) or after (post-EMT) their induction of EMT by TGF-β₁ (5 ng/ml, 24 hours), and the resulting immunocomplexes were reciprocally immunoblotted for β₃ integrin or TβR-II. Direct immunoblot analysis of an aliquot of the prepared cell extracts (Input) served to monitor the total levels of TβR-II, β₃ integrin, FAK, and β-actin. (c) β₃ integrin was immunoprecipitated (β₃ I.P.) from control (scram) and FAK-deficient (shFAK) 4T1 cell extracts, and the resulting immunocomplexes were immunoblotted for TβR-II and FAK, as indicated. Direct immunoblot analysis of an aliquot of the prepared cell extracts (Input) served to monitor the total levels of FAK, TβR-II, and β-actin. Data are representative images from a single experiment that was repeated 4 times and show that FAK deficiency inhibits the formation of β₃ integrin:TβR-II complexes. (d) Grb2 was immunoprecipitated (Grb2 I.P.) from vector control (cntrl), TβR-II- (TβR-II), and FAK-deficient and TβR-II-expressing (TβR-II/shFAK) 4T1 cell extracts, and the resulting immunocomplexes were immunoblotted for TβR-II, FAK, and Grb2, as indicated. Direct immunoblot analysis of an aliquot of the prepared cell extracts (Input) served to monitor the total levels of TβR-II, FAK, and β-actin. Data are representative images from a single experiment that was repeated 3 times and show that FAK deficiency inhibits the interaction between Grb2 and TβR-II. (e) β₃ integrin was immunoprecipitated (β₃ I.P.) from unstimulated (NS) cell extracts derived from 4T1 cells and cells incubated for 18 hours with the FAK inhibitors PF-562271 (271) or PF-573228 (228), as indicated. The resulting immunocomplexes were immunoblotted for TβR-II. Direct immunoblot analysis of an aliquot of the prepared cell extracts (Input) served to monitor the levels of FAK phosphorylated at Y397 (pFAK), total FAK (tFAK), TβR-II, and β-actin. Data are representative images from a single experiment that was repeated 3 times and show that FAK PTK activity is required for the formation of β₃ integrin:TβR-II complexes.