(A) FAK and EGFR activation after seeding serum-starved KindCtr, TlnKo and KindKo cells on PLL or FN and treating them with or without EGF and Mn2+. (B) Immunofluorescence staining of activated (Tyr-397 phosphorylated) FAK and F-actin in cells seeded on FN and treated with Mn2+ for 30 min (FAKGFP indicates exogenous expression of FAKGFP fusion protein). (C) FAK and Akt activation in KindKo cells stably transduced with K2GFP or K2ΔPHGFP either seeded on FN or kept in suspension. GFP indicates similar expression of transduced GFP-tagged constructs. GAPDH levels served to control loading. (D) Levels of phosphorylated signaling mediators downstream of FAK in Mn2+-treated, serum-starved or EGF-treated KindCtr, TlnKo and KindKo cells. GAPDH levels served to control loading. (E) Quantification of lamellipodia formation of FN-seeded TlnKo and KindKo cells treated with Mn2+ and either DMSO or the FAK inhibitor PF-228 (n=3 independent repeats; >100 cells/condition; error bars indicate standard error of the mean; significances are given in comparison to the DMSO control). (F) FAK activity in TlnKo and KindKo cells stably transduced with FAKGFP (n=3 independent experiments). (G) Quantification of lamellipodia formation in TlnKo and KindKo cells stably transduced with FAKGFP (n=3 independent experiments; significances are given in comparison to untreated control; error bars indicate standard error of the mean). Bar, 10 µm. DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FAK, focal adhesion kinase; FAKGFP, green fluorescent protein-tagged FAK; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PLL, poly-L-lysine.
DOI:
http://dx.doi.org/10.7554/eLife.10130.023