Rac3 is a candidate Deaf-1 target gene. Expression of Deaf-1 in whole cell lysates from cells infected with a Deaf-1 retrovirus (+) or control retrovirus (-). Two independent clones (C1 and E1) were generated and used for these studies. Western blots were probed with anti-Deaf-1 antibody. B) Clones transduced with either a Deaf-1 or control retrovirus were plated on coverslips and immunostained with anti-Deaf-1 polyclonal antisera, followed by FITC-conjugated anti-rabbit antibody. Cells were counterstained with DAPI to visualize nuclei by fluorescence microscopy (clones C1 and E1 are shown). C) Quantitative RT-PCR analysis was performed on cDNA generated from Affymetrix RNA samples using primers specific for Deaf-1, Rac3 and 18S rRNA. Data are from two independent experiments and error bars represent the standard deviation from the mean. D) Semi-quantitative RT-PCR analysis of RNA prepared from wild-type or Deaf-1 transgenic mammary glands of 8 week-old mice using primers specific for Rac3 and HPRT.