Persistence of SLP76 microclusters is partially dependent on 14-3-3 proteins binding to phosphorylated Ser376. (A) J14-SLP76-YFP cells were activated for 3 min on anti-CD3–coated coverslips, fixed, and stained with anti-SLP76 and anti–14-3-3ζ antibodies. SLP76-YFP microclusters (left; green in right image) and SLP76–14-3-3ζ complexes detected by in situ PLA (middle; red in right image) were visualized by confocal microscopy. A z-stack projection is shown. Bottom image represents an x-z projection of the cells shown in top images. Images are representative of three independent experiments. Bars, 5 µm. (B) J14 cells were transiently transfected with SLP76-YFP-WT or -S376A constructs. Cells were activated as in A, fixed, and imaged by confocal microscopy. SLP76-YFP microclusters were automatically counted (see Materials and methods) in cells expressing comparable levels of YFP labeling. Horizontal lines and error bars show means and SEM. (C) J14-SLP76-WT cells stably expressing FLAG-SLP76 were stimulated with anti-CD3 mAbs for the indicated time points, lysed, and immunoprecipitated (IP) with anti-FLAG mAbs. Samples were sequentially analyzed by GST (left) and GST–14-3-3ζ (middle) overlay assays followed by immunoblotting with anti-SLP76 and anti-GADS antibodies (right). Similar results were obtained in four independent experiments. Open arrowheads indicate heavy chains of precipitating antibodies. Mobility of molecular mass markers (in kilodaltons) is shown on the left. (D) Human CD4⁺ T cells were treated as in C, immunoprecipitated with anti-SLP76 antibodies, and subjected to overlay assays and immunoblotting as in C. This result is representative of two independent experiments. Mobility of molecular mass markers is shown on the right in kilodaltons.