Fig. 1. Rac isoform expression in bone marrow-derived macrophages (BMMs). (A) RT-PCR to establish primer specificity. RT-PCR was performed on plasmid DNA containing Rac1, Rac2 or Rac3 cDNA using the Rac1/2/3 isoform-specific primers as described in Materials and Methods. (B) RT-PCR to establish Rac isoform expression in BMMs. RT-PCR was performed on cDNA derived from Wt BMM RNA using the same primer sets as in A). As a control for the presence of genomic DNA in the cDNA preparation RT-PCR was also carried out on cDNA prepared in the absence of reverse transcriptase. (C) Analysis of Rac1 and Rac2 protein expression levels in BMMs. Lysate from Wt BMMs and recombinant Rac1 (top) and Rac2 (bottom) protein (lanes 1 and 2; 1.5 and 2.5 ng, respectively) were resolved by SDS-PAGE, western blotted and probed with anti-Rac1 (top) or anti-Rac2 (bottom) antibody. Blots were re-probed with anti-ß-tubulin antibody. (D) Bands on autoradiographs of three western blots similar to those in C were quantified using Kinetic Imaging software. The level of Rac1 or Rac2 protein in BMMs was compared with the level of recombinant Rac1 or Rac2 (lane 1, 1.5 ng).