Figure 5.
Figure 5. HPK1-dependent binding of 14-3-3ζ to GADS. (A) HPK1-HA constructs, wild type (WT) or kinase deficient (KD), were transfected in COS7 cells and immunoprecipitated (IP) with anti-HA mAbs. Recombinant MBP or MBP-GADS was added to either sample and incubated with [32P]ATP. Supernatants were then spotted onto Whatman filters, and incorporated radioactivity was measured. Comparable results have been obtained in three independent experiments. Error bars indicate SDs of triplicates. (B) J14-SLP76-WT cells expressing FLAG-SLP76 and transfected with control (siCont) or HPK1-specific (siHPK1) siRNAs were stimulated by anti-CD3 mAbs for the indicated time points, lysed, and immunoprecipitated with anti-FLAG mAbs. Samples were analyzed by GST (top) and GST–14-3-3ζ (middle) overlay assay followed by Western blotting with anti-SLP76 and anti-GADS antibodies (bottom). Similar results have been obtained in three independent experiments. Open arrowheads indicate heavy chains of the precipitating antibodies. HPK1 knockdown efficiency was assessed by immunoblotting (Fig. S1 C). Mobility of molecular mass markers is shown on the right in kilodaltons. (C) Quantification of GST–14-3-3ζ binding to SLP76 (left) and GADS (right) in overlay assays shown in B. Bands in B (middle and bottom) were acquired and quantified as outlined in Materials and methods. Intensity of bound GST–14-3-3ζ bands was normalized by the relative amount of SLP76 (left) or GADS (right) in the same lane. (D) Jurkat cells transfected with control or HPK1-specific siRNAs were activated for 10 min on anti-CD3–coated coverslips, fixed, and stained with the antibody pairs indicated on the left and by DAPI for visualizing nuclei (blue). Protein complexes (red) were detected by in situ PLA. A z-stack projection is shown in each image. Bars, 5 µm. (E) Images obtained by in situ PLA experiments as shown in D were analyzed using the BlobFinder software to automatically count spots generated by protein–protein interactions. Histograms show means and SEM of the number of spots per cell from two independent experiments. The number of cells analyzed is indicated beside each bar.

HPK1-dependent binding of 14-3-3ζ to GADS. (A) HPK1-HA constructs, wild type (WT) or kinase deficient (KD), were transfected in COS7 cells and immunoprecipitated (IP) with anti-HA mAbs. Recombinant MBP or MBP-GADS was added to either sample and incubated with [32P]ATP. Supernatants were then spotted onto Whatman filters, and incorporated radioactivity was measured. Comparable results have been obtained in three independent experiments. Error bars indicate SDs of triplicates. (B) J14-SLP76-WT cells expressing FLAG-SLP76 and transfected with control (siCont) or HPK1-specific (siHPK1) siRNAs were stimulated by anti-CD3 mAbs for the indicated time points, lysed, and immunoprecipitated with anti-FLAG mAbs. Samples were analyzed by GST (top) and GST–14-3-3ζ (middle) overlay assay followed by Western blotting with anti-SLP76 and anti-GADS antibodies (bottom). Similar results have been obtained in three independent experiments. Open arrowheads indicate heavy chains of the precipitating antibodies. HPK1 knockdown efficiency was assessed by immunoblotting (Fig. S1 C). Mobility of molecular mass markers is shown on the right in kilodaltons. (C) Quantification of GST–14-3-3ζ binding to SLP76 (left) and GADS (right) in overlay assays shown in B. Bands in B (middle and bottom) were acquired and quantified as outlined in Materials and methods. Intensity of bound GST–14-3-3ζ bands was normalized by the relative amount of SLP76 (left) or GADS (right) in the same lane. (D) Jurkat cells transfected with control or HPK1-specific siRNAs were activated for 10 min on anti-CD3–coated coverslips, fixed, and stained with the antibody pairs indicated on the left and by DAPI for visualizing nuclei (blue). Protein complexes (red) were detected by in situ PLA. A z-stack projection is shown in each image. Bars, 5 µm. (E) Images obtained by in situ PLA experiments as shown in D were analyzed using the BlobFinder software to automatically count spots generated by protein–protein interactions. Histograms show means and SEM of the number of spots per cell from two independent experiments. The number of cells analyzed is indicated beside each bar.

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