LAT is required for tyrosine phosphorylation of PLCγ-1 and SLP-76 on CD2 stimulation of Jurkat T cells.
The LAT-deficient Jurkat T-cell line ANJ3 and the ANJ3 reconstituted with wild-type LAT (ANJ3-LATwt) (107 cells/test) were either left unstimulated or stimulated at 37°C for 5 minutes via either the CD2 or TCR-CD3 molecule, as indicated in Figure 1, and lysed in 1% Brij97 lysis buffer. PNLs were immunoprecipitated with 20 μL of antiphosphotyrosine (PY99) mAb-agarose conjugated, separated by SDS-PAGE, and immunoblotted with 4G10 (upper panel). The same membrane was stripped and blotted with anti-PLCγ-1 and anti-SLP-76 antibody (lower panels). The bands corresponding to PLCγ-1, SLP-76, ZAP70, LAT, and zeta-chain proteins are indicated. Results are representative of 2 independent experiments.
