Figure 1.

T cells from transgenic mice express activated PKB. (A) Illustration of the gag-PKB transgene. A 2.3-kb cDNA fragment containing the coding sequence of gag-PKB was inserted into the EcoRI and SmaI sites of the human CD2 minigene. The CD2 promoter, polyadenylation signal (Poly A), and enhancer/locus control region (LCR) are indicated. The construct was linearized at the KpnI and XbaI sites before microinjection. (B) gag-PKB T cells express transgenic gag-PKB mRNA. DNase-treated total RNA isolated from gag-PKB transgenic (B6/PKB) and nontransgenic (B6) LN T cells was subjected to RT-PCR using gag-PKB–specific primers. PCR was performed on the RNA to control for DNA contamination. In the right lane, gag-PKB cDNA was amplified as a positive control for the PCR reaction. RT-PCR products were analyzed on a 1% agarose gel stained with ethidium bromide. (C) Transgenic gag-PKB T cells display elevated levels of PKB protein and increased levels of PKB phosphorylation. Expression of PKB was assessed in thymus, LN, and purified CD4⁺ and CD8⁺ T cells from wild-type mice (B6) and gag-PKB transgenic mice from two independent founder lines (B6/PKB^5-4 and B6/PKB^4-1) by Western blot analysis with a polyclonal anti-PKB antibody. Phosphorylated PKB was detected for the same samples using a polyclonal antibody specific for PKB phosphorylated at Ser-473. Lysates from 2 × 10⁶ cells per tissue sample were loaded per lane after normalization for protein content via the Bradford assay.