FIG. 1.

Expression of Prep1 during T-cell development in wild-type and Prep1^i/i mice. (A) RT-PCR analysis of sorted subpopulations of wild-type thymocytes. Total RNA was isolated from DN, DP, CD4⁺ (CD4), and CD8⁺ (CD8) cells. Serial dilutions (1:5) of cDNA were amplified with Prep1 and β-actin primers (see Materials and Methods). (B) Relative amounts of the Prep1 PCR product are shown in the histogram. (C) Nuclear (N) and cytoplasmic (C) extracts of Prep1^i/i (i/i) and their wild-type controls (+/+) were blotted onto PVDF membranes and incubated with anti-Prep1 antibody. Nuclear extract from a Prep1-overexpressing cell line (F9) (30) was used as a positive control. Notice the overexposure of the Prep1 row (middle blot) to check for possible Prep1 protein. Anti-Sp1 and anti-β-actin antibodies were used as loading controls for nuclear and cytoplasmic extracts. (D) RT-PCR analysis of Prep1 expression. RNA was isolated from total thymocytes from two Prep1^i/i mice (i/i) and their wild-type control littermates (+/+). In the case of Prep1^i/i mRNA, 32 cycles of PCR were performed, while for β-actin mRNA, only 24 cycles were performed. The no-RT control shows the absence of product in a parallel reaction without the addition of reverse transcriptase. Primers for β-actin were used as a control.