Expression and localization of 4.1R in mouse CD4⁺ T cells. (A) Exon composition of 4.1R isoforms. Schematic representation of the exon map of 4.1R is displayed in the top panel. Two translation initiation sites are indicated. Alternatively spliced exons are shown in black, constitutive exon in gray, and noncoding exons in open boxes. Exon compositions of 4.1R 135 kDa and 80 kDa are shown in the middle and bottom panels, respectively. (B) Western blot analysis of protein 4.1 members. CD4⁺ T cells (10⁶ cells) purified from 4.1R^+/+ or 4.1R^−/− T lymph nodes were subjected to immunoblot analysis with polyclonal rabbit antibodies against 4.1R exon 13 (i), 4.1B headpiece (ii), 4.1G headpiece (iii), and 4.1N headpiece (iv). The positions of approximately 135 kDa and approximately 80 kDa 4.1R are indicated. Note that, although 4.1B and 4.1G are unchanged, 4.1N expression is up-regulated in 4.1R^−/− CD4⁺ T cells. (C) Localization of 4.1R in T cells. Unstimulated and stimulated primary mouse T cells were doubled stained with anti-4.1R exon 13 and anti-LAT antibodies and analyzed by confocal microscopy. In unstimulated samples, protein 4.1R is evenly distributed in discrete puncta around the cell membrane where it dose not colocalize with LAT. After stimulation, both protein 4.1R and LAT become enriched at the interface between T cell and bead where they colocalize. Scale bar, 2 μm.