Characterization of platelet IQGAP2.
(A) Schema of the IQGAP2/PAR gene cluster integrating consensus data from Celera18 and public databases19 with those generated by PCR, restriction analysis, and sequence analysis. Approximate distances in kilobases are outlined, as are selected restriction enzymes used for genomic mapping (E indicatesEcoRI; X, XbaI). Exons are depicted as solid boxes, except for IQGAP2 exon 1 (fine stipples), which has not been identified within either database, and PAR3 exons (cross-hatches) (cen, centromere; tel, telomere). (B) Southern blot analysis using 100 ng YAC798D11 DNA and an IQGAP2 exon 12-specific probe or the PAR3 cDNA as probe. Lane 1 isEcoRI-digested, and lane 2 is XbaI-digested. Note that PAR3 exon 2 and IQGAP2 exon 12 are contained within the identical EcoRI fragment. (C) PCR using 100 ng cosmid 3-2 DNA and primer pair G18F/P01F (lane 1) or P02R/G15R (lane 2). DNA sequence analysis confirmed the identity of the PCR fragments. Fragment sizes corresponding to HindIII-digested bacteriophage λ DNA are shown in panels B and C. (D) Platelet immunoprecipitations were completed using 1 × 108RIPA-solubilized gel-filtered platelets (GFP), and anti-IQGAP2 monoclonal antibody (mAb) (lane 2) or preimmune mouse IgG (lane 3), followed by immunoblot analysis using the IQGAP2-specific mAb (arrow). The corresponding SDS-solubilized GFP lysate is shown in lane 1.
