Deficient TCR triggering in CD4⁺ cells lacking Cav-1. (A) Purified conventional CD4⁺ T cells were incubated for 16 hours in the presence of the indicated amounts of plate-bound anti-TCR (145-2C11) antibodies. Cells were harvested, stained with antibodies against CD4 and CD69, and analyzed by flow cytometry. Results are representative of 4 independently performed experiments. (B) Purified conventional CD4⁺ T cells were stimulated with the indicated amounts of anti-TCR antibodies for 5 minutes. Cells were fixed, intracellularly stained with an antibody against phospho-ERK1/2, and analyzed by flow cytometry. Two independently performed experiments are shown pooled in the left panel. (C) Purified CD4⁺ T cells were stimulated with 3 μg/mL anti-TCR (145-2C11) antibody for 5 minutes. After cell lysis, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by WB for anti–phospho-ζ (Y-142), anti–phospho-ε, and anti-GAPDH (1 of 3 comparable experiments is shown). P-ERK, phospho-extracellular signal-regulated kinase.