HPK1 overexpression affects SLP76 microclusters. (A) J14-SLP76-YFP cells were transfected with HPK1-mCherry. The day after transfection, cells were activated for 3 min on anti-CD3–coated coverslips, fixed, and analyzed by confocal microscopy. A single optical section at the level of the contact surface is shown. The plot shows the number of microclusters in cells expressing or not expressing the HPK1-mCherry construct. (B) J14-SLP76-YFP cells were transfected with HPK1-KD-mCherry and analyzed as in A. (C) J14-SLP76-YFP cells were transfected with HPK1-Y379F-mCherry and analyzed as in A. For all panels, images are representative of more than three independent experiments. Horizontal lines and error bars show means and SEM. Bars, 5 µm.