Fig. 7. Dimerization and the SH3 domain of ßPIX are required for the recruitment of GIT1 at large cytoplasmic structures. (A) Schematic diagram of the PIX constructs used in this study; SH3, Src homology 3 domain; DH, Dbl homology region; PH, pleckstrin homology domain; ABD, ArfGAP binding domain; LZ, leucine zipper. (B-D) Effects of ßPIX expression on GIT1 localization. CEFs were transfected with either GIT1 or PIX (B), cotransfected with both proteins (C), or cotransfected with full length GIT1 and the monomeric SH3 mutant ßPIX-PGLZ (D). Bar, 20 µm. (E) Quantification of the localization of GIT1 at large cytoplasmic structures in CEFs transfected with GIT1 alone (n=300); cotransfected with GIT1 together with each of the following ßPIX constructs: full length PIX (n=385), PIX-PGLZ (n=200), PIXLZ (228), PIX-PG (200), or cotransfected with the monomeric mutant of GIT1 (p95-LZ) and full length PIX (n=200). Error bars represent the s.d. from at least two independent experiments. Statistical significance was assessed by the Student's t-test (P<0.05 considered significant). The values from cells transfected with GIT1 alone or cotransfected with GIT1 and each of the indicated ßPIX mutants were compared to the values from cells cotransfected with GIT1 and wild-type ßPIX. *P<0.005; **P<0.0005. (F) Overexpression of either PIXLZ or PIX-PGLZ alone did not induce the formation of large structures. Bar, 20 µm. (G) Immunoprecipitation with anti-FLAG from lysates of COS7 cells transfected with FLAG-GIT1 and one of the following monomeric mutants of ßPIX: ßPIX-PGLZ (IP1), ßPIX-CLZ (IP2), ßPIX-LZ (IP3), ßPIX-PHLZ (IP4) and ßPIXwt (IP5). 50 µg of each lysate (Lys) were also loaded. Portions of the same filters were immunoblotted for PIX and GIT1, as indicated.