Fig. 1. Induction of CD44 variant expression by anti-CD3ε/anti-CD28 antibodies in murine primary T cells is repressed by inhibition of the ERK pathway. (A) Simplified scheme of the murine CD44 gene organization indicating the positions of PCR primers (arrows) used for exon-specific RT–PCR. Open boxes represent constant exons, filled boxes denote variant exons, thin lines represent intervening intron sequences. (B–F) Exon-specific RT–PCR analysis of CD44 variant expression. Murine primary T cells were either left untreated (B), induced with TPA (40 ng/ml) (C) or stimulated by anti-CD3ε/anti-CD28 antibodies coated to plates for 6 h (D–F). In addition, cells were treated either with the solvent dimethylsulfoxide (DMSO) (D), the MEK inhibitor U0126 (E) or the p38 inhibitor SB203580 (F). For exon-specific RT–PCR 5′ primers hybridizing to either the 5′ constant region (C13, lane 1) or to the variant exons (lanes 2–11) were used together with a primer (C2A) recognizing the 3′ constant region. Asterisks mark an unspecific PCR product (König et al., 1996). Identity of bands in lanes 5, 6 and 11 was confirmed by sequencing. The lower band in (D) lane 11 represented a PCR artifact that was only seen in some experiments. (G) Activation of ERK and p38 MAP-kinase pathways: phosphorylation of ERK 1/2 (p42/p44) and p38 was examined by western blotting with phosphospecific antibodies after pre-treatment with U0126 or SB203580 for 15 min and subsequent induction with anti-CD3ε/anti-CD28 antibodies for 10 min. (H) Activation of the JNK MAP-kinase pathway: phosphorylation of JNK (arrow) was examined by western blotting with an anti-phospho-JNK antibody 10 min, 1 h, 2 h and 6 h after induction with anti-CD3ε/anti-CD28 or after treatment with TPA (40 ng/ml) for 10 min as a positive control.