Paxillin regulates HDAC6 activity through its proline-rich region. (A) Schematic representation of the domain structure of paxillin. The proline-rich region (Pro) is demarcated in red and spans amino acids 46–56, which are deleted in the GFP-paxillinΔPro construct. N, N terminus; C, C terminus. (B and C) Images of paxillin^−/− MEFs transfected with GFP-tagged paxillin constructs as indicated. Cells were plated on fibronectin-coated coverslips 16 h before fixation, immunofluorescence staining, and relative acetylated α-tubulin MFI quantitation (C). MFI was quantified from a minimum of 17 cells from three individual experiments. (D) Western blots of GFP-paxillin co-IP with endogenous HDAC6 in paxillin^−/− MEFs. WB, Western blot. Equal protein input was determined by Western blotting of the respective detergent soluble lysates (DSL). (E and F) Images (E) and quantitation (F) of PLA spots between the GFP-tagged paxillin constructs as indicated and endogenous HDAC6. Boxed regions indicate the area used for the respective zoomed images. Data are represented as the mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.