Figure 1.

Rac1 deletion in MEF cultures. (A) Flow cytometry analysis of EGFP expression. A floxed EGFP reporter gene is activated by cre-mediated recombination in these cells (see Materials and Methods). Cells with genotypes Rac1^k/c (k/c), and Rac1^w/c (w/c) are primary cultures, whereas cells with genotype Rac1^c/c (c/c) are immortalized. Note that nearly all cells contain EGFP after HTNC exposure. (B) Southern blot analysis of DNA from immortalized (c/c) and primary (w/c and k/c) MEF cultures was digested with StuI and probed with a DNA fragment from Rac1 intron 1. Bands corresponding to different alleles of Rac1 are indicated, conditional (c) 11 kb, knockout (k) 7 kb, and wild-type (w) 5 kb. Note that conversion of the c to the k allele is qualitatively complete after HTNC treatment. (C) Western blot analysis showing Rac1 depletion after HTNC treatment, using an antibody that recognizes all three Rac isoforms (see Supplementary Figure S1). Tubulin serves as a loading control. 1×, 5×, etc. indicate relative amount of cell lysate loaded. The genotypes of cells are indicated, k/c-k/k means Rac1^k/c converted to Rac1^k/k, etc. All data are shown for cells 4-5 d after HTNC application.