Figure 3

Expression and binding to α4 of paxillin mutant isoforms. A) Schematic representation of paxillin, showing the position of the 3 phosphorylated tyrosine residues, the LD domains and the LIM domains. B) The three constructs used to examine paxillin function; wild-type paxillin (paxwt), Tyr to Phe mutations of the 3 tyrosines (pax3Y) and an LD4 domain deletion (paxLD4). All three contain a FLAG tag. C) Western blotting of lysates from PC12 cells expressing the different paxillin constructs. All three are expressed at a similar level, but the pax3Y mutant is not recognised by the anti-phosphotyrosine PY20 antibody. D) Western blots using anti-FLAG tag and anti-paxillin antibodies of lysates from PC12 cells expressing the different paxillin constructs with or without co-expression of wild-type α4 integrin, and from mock-transfected PC12 cells. The anti-FLAG (top panel) recognises only expressed paxillin, while the anti-paxillin antibody (middle panel) recognises both expressed and endogenous paxillin. Note that the normal endogenous levels of paxillin (seen in the mock-transfected cells with the anti-paxillin antibody) are lower than those seen following expression of the three constructs, with or without α4 (middle panel), and that all cell lines contain similar levels of expressed paxillin (top panel). A β-actin loading control is also shown in the lower panel. E) Lysates of biotin-labelled PC12 cells expressing either wild-type α4 alone, wild-type paxillin alone or wild-type α4 with the different paxillin constructs immunoprecipitated with anti-α4 as in Fig 1 and then re-immunoprecipitated (Re-IP) with anti-FLAG antibodies before western blotting with anti-paxillin antibody (top panel) or visualised by streptavidin-peroxidase/ECL (lower panel). All four cell lines transfected with α4 constructs express the integrin at similar levels (lower panel), and all expressed (wild-type and mutant) paxillins bind to the integrin equally well (upper panel). In the absence of any α4 integrin (wtpax only in upper panel), no expressed paxillin is detected, confirming the specificity of the assay. Note that the anti-FLAG antibody used to detect paxillin would not be expected to detect the endogenous paxillin associated with α4, and so no band is seen in the α4-only lane of the upper panel.