Figure 1.

Decreased Rxra expression in thymocytes and T lymphocytes from cell-specific knockout mice. (a) PCR analysis of genomic DNA confirms floxing-out of exon 4 of the Rxra gene in thymocytes and T lymphocytes but not B lymphocytes from lck-Cre mice with floxed RXR-α allele. Thymocytes from one homozygous (RXR-α^ko/ko) and one heterozygous (RXR-α^wt/ko) mouse were sorted into pools of single-positive (CD4, CD8) and double-positive (DP) cells. Splenocytes from the same mice were sorted into pools of CD4⁺ T lymphocytes (CD4; CD4⁺ CD3⁺), CD8⁺ T lymphocytes (CD8; CD8⁺ CD3⁺) and B lymphocytes (B; B220⁺ CD3^–). As described in the Materials and methods, the floxed (flox), wild-type (wt) and floxed-out (fo) RXR-α alleles were identified with PCR primers (P1 and P3) flanking the floxed region of Rxra. Data are representative of six homozygous and six heterozygous mice sorted in this fashion. Molecular size standards (in base pairs) are shown at the far left. (b) Western blot analysis confirms decreased RXR-α expression in thymocytes and T lymphocytes from knockout mice. Whole-cell protein extracts (10 μg/lane) of total thymocytes (Th), T lymphocytes isolated from splenocytes with Thy-1.2-selective magnetic beads (+), and Thy-1.2-negative splenocytes (–) were analysed by Western blot using an RXR-α-specific antibody. Cell extracts were from one homozygous (RXR-α^ko/ko) and one wild-type (RXR-α^wt/wt) mouse. A nuclear protein extract (10 μg) from F9 (F9) embryocarcinoma cells was included as a positive control for RXR-α, which runs at approximately 54 000 and is indicate by an asterisk (*). Results are representative of blots from six homozygous and five wild-type mice. Molecular size standards are indicated at left. (c) Bar graph showing mean + SE of the intensity of staining for RXR-α by Western blot of Thy-1.2-positive splenic T lymphocytes from six homozygous and five wild-type mice, and from unselected thymocytes of two homozygous and two wild-type mice. The means differed between genotypes at *P =0·013 using paired Student's t-test. Samples of the same cell type from the same blot (which contained the same amount of protein per lane) were paired for analysis. One blot contained T-lymphocyte samples from two homozygous mice and one wild-type mouse and the band intensities for the homozygous knockout mice were averaged for statistical analysis.