Biochemical analysis of signal transduction pathways in 4.1R^+/+ and 4.1R^−/− CD4⁺ T cells. (A) Western blot analysis of signal molecules. 4.1R^+/+ or 4.1R^−/− T cells were stimulated with anti-CD3ϵ (5 μg/mL) for various time periods, and indicated proteins were immunoblotted for the level of expression and for the extent of phosphorylation. Note the significantly enhanced phosphorylation of LAT and ERK in 4.1R^−/− T cells. (B) Quantitative analysis of pLAT and pERK. The phosphorylation levels of LAT or ERK after 15 minutes of stimulation were quantified with the use of ImageJ software (National Institutes of Health, Bethesda, MD). Note the significantly increased phosphorylation of both LAT and ERK. (C,D) Intracellular staining of pERK. Intracellular staining of pERK was described in “Intracellular staining.” The representative profiles of pERK of 4.1R^+/+ and 4.1R^−/− CD4⁺ T cells are shown (C), and the quantitative analysis is shown (D). Note a significantly higher expression of pERK in 4.1R^−/− CD4⁺ T cells. (E) Western blot analysis of phosphor-residues of LAT. 4.1R^+/+ or 4.1R^−/− T cells were stimulated with anti-CD3ϵ (5 μg/mL) for 15 minutes, and Western blot analysis was performed using anti-LAT phosphor-residue–specific antibodies as indicated. Note the significantly enhanced phosphorylation of Y175 and Y195 but not Y132 and Y235 of LAT in 4.1R^−/− T cells. Error bars indicate SD.