(
A) Immunofluorescence images of REF52 stained for FAK pY407, paxillin pY118, and CAS pY165 using phospho-specific antibodies. Scale bars, 15 μm. (
B) A REF52 cell co-expressing meGFP-dSH2 and TD-mKate2-paxillin. Note the localization of dSH2 in cell-matrix adhesion sites, as previously reported (
Kirchner et al., 2003). Scale bar, 10 μm. (
C) Top and middle rows, FLIM images color-coding the fraction (α) of the donor- (mCitrine) tagged protein (FAK, paxillin, CAS, or ILK) that FRET to the acceptor- (mCherry) tagged dSH2 before and after acceptor photobleaching. ILK is used here as a negative control for tyrosine phosphorylation. Note that low levels of α are observed also for ILK in focal adhesions, plausibly due to density effects. However, FAK, paxillin, and CAS exhibit higher α values, suggesting FRET due to direct interaction. Scale bars, 10 μm.