TG CD4⁺ lymphocytes display less NF-κB transcriptional activity. CD4⁺ cells from TG and WT mice were transfected with pRL-TK in combination with either (A) pGL3-TK-kBPD or (C) AP-1–Luc reporter plasmid. After 24 hours, cells were stimulated with anti-CD3/anti-CD28 Abs for the indicated times and luciferase activity was assessed. Results are expressed as fold induction (mean ± 1 SEM of 3 independent experiments) of the sample incubated with anti-CD3/anti-CD28 Abs versus the corresponding untreated sample, with the control value being 1. ^*P < .05; ^**P < .01. (B) Immunofluorescence analysis of NF-κB in TG and WT CD4⁺ cells activated with anti-CD3/anti-CD28 Abs for 6 hours, and stained with anti-p65 Ab by paraformaldehyde-saponin procedure. Images were captured with a Leitz Dialux 20 microscope using a 100×/1.25 NA oil objective. (D) ERK phosphorylation was evaluated in CD4⁺ cells stimulated for 15 or 30 minutes with anti-CD3/anti-CD28 Abs. Whole-cell lysates were probed with anti-phospho–ERK-1/2 (pERK-1/2) or with anti–ERK-1/2 Abs.