PLCγ1 cross-links LAT by two SH2 domains. (A) Domains of the proteins used in the study. (B) TIRF microscopy revealed that both nSH2 and cSH2 are required for PLCγ1-driven LAT microcluster formation. SH3 domain promotes cluster formation. Alexa Fluor 488–labeled LAT at 300 molecules/µm² was incubated with 300 nM Sos1 and 50 nM PLCγ1 for 0.5 h before imaging. Scale bar, 5 µm. (C) Quantification of PLCγ1-driven LAT microclusters. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05. (D) Schematics of the assay of testing SH2 domain binding sites. (E) PLCγ1 nSH2 binds LAT Y132. Phosphopeptides were synthesized, biotinylated at the N terminus, and attached to the biotin-functionalized supported lipid bilayers by streptavidin. The SH2 domains were labeled with fluorescent dye (Maleimide-Ax647) and incubated with the individual phosphopeptides. The membrane-associated SH2 domain was measured by TIRF microscopy. Scale bar, 5 µm. (F) PLCγ1 cSH2 binds LAT Y171. Same settings as in E.