HPK1 uncouples SLP76 from LAT microclusters. (A) J14-SLP76-YFP cells transfected with control (siCont) or HPK1-specific (siHPK1) siRNAs were activated for 3 min on anti-CD3–coated coverslips, fixed, stained with antiphospho-LAT (Y191) mAbs, and imaged by confocal microscopy. Results are representative of three independent experiments. (B and C) J14 cells expressing SLP76-YFP were transfected as in A and stimulated for the indicated time points. SLP76-YFP (B) and phospho-LAT (p-LAT; C) microclusters were imaged and automatically counted (see Materials and methods). Circles represent the number of microclusters/cell of pooled data from three independent experiments. (D) Human primary CD4⁺ T cells, transfected with control or HPK1-specific siRNAs, were activated as in A, fixed, and stained with antiphospho-SLP76 (Y128) and antiphospho-LAT (Y191) mAbs. Images were acquired as in A. HPK1 knockdown efficiency was assessed by flow cytometry (Fig. S2 B). (E and F) Human primary CD4⁺ T cells were transfected and stimulated as in D and fixed at the indicated time points. Phospho-SLP76 (p-SLP76) and phospho-LAT microclusters were counted as in B. (G) J14-SLP76-WT cells expressing FLAG-SLP76 and transfected with control or HPK1-specific siRNAs were stimulated by anti-CD3 antibodies for the indicated time points, lysed, and immunoprecipitated (IP) by the anti-FLAG mAb. Proteins were separated by electrophoresis in nonreducing conditions and analyzed by immunoblotting (IB) as indicated. Comparable results have been obtained in two independent experiments. See Fig. S3 A for analysis of HPK1 knockdown efficiency. (H) J14 cells transiently transfected with SLP76-FLAG and/or HPK1-MYC constructs were stimulated for the indicated time points, lysed, and immunoprecipitated by anti-FLAG antibodies. Proteins were analyzed by immunoblotting as in G. Mobility of molecular mass markers (in kilodaltons) is shown on the left. Horizontal lines and error bars show means and SEM. Bars, 5 µm.