Endogenous paxillin and HDAC6 exhibit a direct interaction throughout the cell. (A and B) Representative images (A) and quantitation (B) of a proximity ligation assay (PLA) performed in MDA-MB-231 cells with protein-specific antibody pairs as indicated. Asterisks highlight cells with the most PLA spots and robust F-actin stress fibers. (C and D) Western blots of endogenous paxillin co-IP with HDAC6 in MDA-MB-231 cells. (E and F) Images of paxillin^−/− MEFs expressing mRFP-paxillin and HDAC6-GFP (E) or mRFP and HDAC6-GFP (F) subjected to apFRET. Dashed lines demarcate hot spots of FRET associated with FAs. (G) Quantitation of the FRET efficiency between FRET pairs as indicated. FRET values were quantified from a minimum of 16 cells and three individual repeats. (H) Images of MDA-MB-231 cells expressing GFP-tagged talin and analyzed using a PLA as indicated. PLA spot colocalization with talin-GFP (arrows) was determined from a minimum of 13 cells from n = 2 individual experiments. Data are represented as the mean ± SEMs. ***, P < 0.0005. Boxed regions indicate the areas used for the zoomed insets and cropped images.