PLCγ1 protects LAT from dephosphorylation by CD45. (A) Reduced phosphorylation at LAT Y132 in PLCγ1-null cells. Cells as indicated were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. The level of indicated proteins, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. MW, molecular weight. (B) PLCγ1 prevents LAT Y132 from being dephosphorylated. Cells as indicated were pretreated with 0.1 mM vanadate (pan-phosphatase inhibitor) before being stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. The level of indicated proteins, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01; ns, not significant. (C) Schematics of the in intro dephosphorylation assay. (D) PLCγ1 prevents LAT Y132 from being dephosphorylated by CD45 in vitro. pLAT, at 1,000 molecules/µm2, was incubated with 1 µM Grb2, 0.5 µM Sos1, and/or 100 nM full-length PLCγ1 for 0.5 h. CD45 was then added to dephosphorylate pLAT for 5 min. The reaction was terminated by adding SDS-PAGE loading buffer with 2 mM vanadate. The level of phosphorylated LAT, after being normalized to total LAT, was quantified. Shown are mean ± SD; n = 3 independent experiments.
