PLCγ1 promotes LAT clustering, SLP76 phosphorylation, and ERK activation in Jurkat T cells. (A) Diminished LAT microcluster formation in PLCγ1-null cells. Wild-type or PLCγ1-null Jurkat T cells expressing LAT-mCherry were plated on OKT3-coated cover glass. LAT microcluster formation was revealed by TIRF microscopy. Images showed clustering 90 s after cell landing on the glass. Scale bar, 5 µm. Shown are mean ± SEM; n = 25 or 26 cells. Unpaired two-tailed t test was used. **, P < 0.01. (B) The nSH2 and SH3 domain of PLCγ1 promotes LAT cluster formation. PLCγ1-null Jurkat T cells expressing LAT-mCherry were reconstituted with the GFP-tagged wild-type, ΔnSH2, or ΔSH3 PLCγ1. Those cells were plated on OKT3-coated cover glass. Images showed clustering 90 s after cell landing on the glass. LAT microcluster formation was revealed by TIRF microscopy. Scale bar, 5 µm. Shown are mean ± SEM; n = 22–30 cells. Unpaired two-tailed t test was used. **, P < 0.01. (C) Immunoblot analysis of LAT-null Jurkat T cells reconstituted with the GFP-tagged wild-type, ΔnSH2, or ΔSH3 PLCγ1. Cells were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. MW, molecular weight. (D) Quantification of the level of indicated proteins, after being normalized to the expression level of GAPDH. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05; **, P < 0.01.