Journal: Brain, behavior, and immunity

Article Title: Functional circuitry of neuro-immune communication in the mesenteric lymph node and spleen.

doi: 10.1016/j.bbi.2019.08.188

Figure Lengend Snippet: Confocal microscopy was conducted on tissue sections ChAT-GFP (C57BL/6 background) mice for T-cells (anti-CD3), ChAT (anti-GFP), and sympathetic axons (anti-TH). The section was identified and overlapping tiles were acquired from 943 µm x 454 µm area of MLN (upper panel, scale bar =100 µm). Areas indicated in the bounding boxes are shown in the middle (CD3, ChAT-GFP, TH) and lower panels (ChAT-GFP, TH, scale bar = 20 μm). Region of the hilus in the MLN is within box “i”. Representative images obtained from 8 mice.

Article Snippet: Primary antibodies used in this study were rabbit Anti-TH (Millipore, AB152, Billerica, MA), goat anti-GFP (Rockland Immunochemicals, Limerick, PA), rabbit anti-TNF (Abeam, AB6671) and rat anti-CD3 (clone CD3-12, Bio-Rad, Hercules, CA).

Techniques: Confocal Microscopy

Journal: The Journal of Experimental Medicine

Article Title: A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

doi: 10.1084/jem.20062066

Figure Lengend Snippet: Detection of inducible association of 14-3-3 proteins to SLP-76 and serine 376 phosphorylation. (A) WT#27 cells overexpressing FLAG–SLP-76 and parental T8.1 cells were left unstimulated (−) or activated by anti-CD3 cross-linking (+) for 5 min at 37°C. Lysates were incubated with anti-FLAG affinity resin, and bound proteins were eluted with an excess of FLAG peptide. Eluates were then fractionated by SDS-PAGE, and proteins were detected by colloidal blue staining. Relevant protein bands were excised, digested in gel by trypins, and identified by MALDI-TOF peptide mass fingerprinting. Migration of protein standards is indicated on the left. (B) WT#27 cells were left unstimulated (ctrl), activated at 37°C by CD3 cross-linking as in A, or treated with 1 μM calyculin A (Cal.A), 10 ng/ml PMA, or 1 μM okadaic acid (OA) for 30 min at 37°C. Lysates were immunoprecipitated with anti-FLAG antibodies as described in A and immunoblotted with anti-FLAG (top) or anti–14-3-3 (bottom) antibodies. (C) FLAG–SLP-76 was immunoprecipitated from CD3 cross-linking–stimulated WT#27 cells as described in A. The protein band was excised and digested in gel by trypsin and AspN. The resulting peptide mixture was analyzed by nanoESI MS. The acquired MS/MS dataset was submitted to sequence tag scanning to spot modified peptides. The MS/MS spectrum shows fragment ions of a peptide spanning residues 376–391 of SLP-76, which clearly demonstrates phosphorylation of S376.

Article Snippet: The following mAbs were used: anti-FLAG M2 and anti-GST (Sigma-Aldrich); anti–SLP-76 (SLP-76/03) and anti–human CD3 (UCHT1; Serotec); anti-HA (F7; Santa Cruz Biotechnology, Inc.); anti-phosphorylated SLP-76 Y113 and Y128 (BD Biosciences); anti-CD28 (CD28.2; Immunotech); and anti–mouse CD3 (145-2C11; reference 40).

Techniques: Incubation, SDS Page, Staining, Peptide Mass Fingerprinting, Migration, Immunoprecipitation, Tandem Mass Spectroscopy, Sequencing, Modification

Journal: The Journal of Experimental Medicine

Article Title: A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

doi: 10.1084/jem.20062066

Figure Lengend Snippet: Serine 376 of SLP-76 controls the interaction with 14-3-3 proteins. (A) COS-7 cells were transfected with a myc–14-3-3ζ–encoding plasmid together with FLAG–SLP-76 constructs, either WT or Ser to Ala mutants. After 36 h, cells were harvested and incubated for 10 min at 37°C with medium alone (−) or 1 μM calyculin A (+). Cell lysates were subjected to anti-FLAG immunoprecipitation, and bound proteins were analyzed by anti-FLAG (top row) and anti–14-3-3ζ immunoblotting (second row from top). Protein expression in all samples was assessed by anti-FLAG and anti–14-3-3ζ immunoblotting on whole cell lysates (two bottom rows). (B) J14-derived clones expressing similar amounts of FLAG-SLP-76-WT (clones C5, D9, and E5) or -S376A (clones E6 and E11) were left unstimulated or activated by anti-CD3 cross-linking for 5 min at 37°C. Samples were lysed, immunoprecipitated with anti-FLAG antibodies (top rows), and subjected to anti-FLAG and 14-3-3ζ immunoblotting. Anti-FLAG and anti–14-3-3ζ immunoblots on whole cell lysates (bottom rows) demonstrate comparable expression of transfected SLP-76 constructs and endogenous 14-3-3ζ. (C) J14 cells stably expressing FLAG-SLP-76-WT (clone C5) or -S376A (clone F3) were left unstimulated (medium) or treated with 10 μg/ml anti-CD3 mAb or 1 μM calyculine A (calyc.A) for 5 min at 37°C. Lysates were then immunoprecipitated with an anti-FLAG mAb. After SDS-PAGE and transfer onto nitrocellulose, proteins were denaturated and renaturated, and then probed with either GST-P3 (top left row; reference ) or a GST–14-3-3ζ fusion protein (top right row). Specific binding was detected by an anti-GST mAb (top rows). Membranes were then stripped and reprobed with an anti–SLP-76 antiserum (M14; bottom rows).

Article Snippet: The following mAbs were used: anti-FLAG M2 and anti-GST (Sigma-Aldrich); anti–SLP-76 (SLP-76/03) and anti–human CD3 (UCHT1; Serotec); anti-HA (F7; Santa Cruz Biotechnology, Inc.); anti-phosphorylated SLP-76 Y113 and Y128 (BD Biosciences); anti-CD28 (CD28.2; Immunotech); and anti–mouse CD3 (145-2C11; reference 40).

Techniques: Transfection, Plasmid Preparation, Construct, Incubation, Immunoprecipitation, Western Blot, Expressing, Derivative Assay, Clone Assay, Stable Transfection, SDS Page, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

doi: 10.1084/jem.20062066

Figure Lengend Snippet: Serine 376 phosphorylation is delayed as compared with tyrosine phosphorylation of SLP-76. (A) Jurkat cells were left unstimulated (ctrl) or incubated with 10 μg/ml anti-CD3 mAbs for the indicated time points. Cells were lysed and immunoprecipitated with an anti–SLP-76 mAb. Samples were then analyzed by Western blotting with anti-pS376 (top left) and anti–14-3-3ζ (middle row, left). Membranes were then stripped and reprobed with an anti–SLP-76 mAb (bottom row, left). Fluorescent immunoblots were acquired and quantified by using an Odyssey scanner. The graph (right) shows the quantification of band intensities in the pS376 (□) and 14-3-3ζ (•) immunoblotting, normalized by the amount of immunoprecipitated SLP-76. (B) Jurkat cells were left unstimulated or stimulated by anti-CD3 mAbs as described in A. SLP-76 phosphorylation was analyzed by immunoblotting with mAb anti-pY128 (top left) and affinity-purified rabbit anti-pS376 antibodies (middle row, left). Membranes were then stripped and reprobed with an anti–SLP-76 mAb (bottom row, left). Quantification of anti-pY128 and anti-pS376 and immunoblots, performed as explained in A, is shown on the right. (C) Human T cell blasts were stimulated with anti-CD3 mAb for the indicated time points. SLP-76 immunoprecipitates were subjected to anti-pY128 (top row), anti-pS376 (second row from top), anti–SLP-76 (third row from top), and anti–14-3-3ζ (bottom row) immunoblotting.

Article Snippet: The following mAbs were used: anti-FLAG M2 and anti-GST (Sigma-Aldrich); anti–SLP-76 (SLP-76/03) and anti–human CD3 (UCHT1; Serotec); anti-HA (F7; Santa Cruz Biotechnology, Inc.); anti-phosphorylated SLP-76 Y113 and Y128 (BD Biosciences); anti-CD28 (CD28.2; Immunotech); and anti–mouse CD3 (145-2C11; reference 40).

Techniques: Incubation, Immunoprecipitation, Western Blot, Affinity Purification

Journal: The Journal of Experimental Medicine

Article Title: A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

doi: 10.1084/jem.20062066

Figure Lengend Snippet: SLP-76–S376A mutant induces higher IL-2 promoter activation and then WT SLP-76. (A) Cells stably expressing FLAG-SLP-76-WT or -S376A mutant (J14-WT or J14-S376A, respectively) were obtained by retroviral infection of J14 cells (see Materials and methods). GFP + cells were then enriched by two round of fluorescence-activated cell sorting to obtain stable cell populations expressing comparable levels of GFP (left). Comparable expression of CD3 (middle) and CD28 (right) by these cells was assessed by flow cytometry using PE-conjugated antibodies. (B) J14, J14-WT, J14-S376A, and Jurkat cells were lysed and analyzed by anti–SLP-76 immunoblotting. (C) J14-WT and J14-S376A cells were transfected with the phIL-2 luciferase reporter. 22 h after transfection, cells were incubated for 6 h with or without increasing amounts of anti-CD3 and 10 μg/ml of soluble anti-CD28. Cell lysis and luciferase activity measurement and normalization are described in Materials and methods. Each point represents average and SD of quadruplicate determination. (D) Aliquot of phIL-2 luciferase–transfected J14-WT and J14-S376A cells (see C) were stimulated with PMA and the Ca 2+ ionophore A23187 to assess maximal IL-2 promoter activation. Luciferase induction was measured as explained above. Histograms and error bars represent average and SD of raw luciferase activity of quadruplicate determinations. RLU, relative luciferase units.

Article Snippet: The following mAbs were used: anti-FLAG M2 and anti-GST (Sigma-Aldrich); anti–SLP-76 (SLP-76/03) and anti–human CD3 (UCHT1; Serotec); anti-HA (F7; Santa Cruz Biotechnology, Inc.); anti-phosphorylated SLP-76 Y113 and Y128 (BD Biosciences); anti-CD28 (CD28.2; Immunotech); and anti–mouse CD3 (145-2C11; reference 40).

Techniques: Mutagenesis, Activation Assay, Stable Transfection, Expressing, Infection, Fluorescence, FACS, Flow Cytometry, Western Blot, Transfection, Luciferase, Incubation, Lysis, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

doi: 10.1084/jem.20062066

Figure Lengend Snippet: Increased SLP-76 and PLC-γ1 tyrosine phosphorylation in T cells expressing the SLP-76–S376A mutant. (A) J14-WT and J14-S376A cells were left unstimulated or stimulated by anti-CD3 mAb for the indicated time points. Lysates were fractionated on NuPAGE gel and immunoblotted with anti–SLP-76 pS376 (top row) and pY128 antibodies (second row from top), followed by anti–PLC-γ1 pY783 antibodies (third row from top). Equal protein loading in each lane was assessed by 14-3-3ζ immunoblotting (fourth row from top). Aliquots of the same lysates were analyzed on a separate gel to check for equal amount of SLP-76 constructs in all samples (bottom row). (B) Graphs show the comparison of SLP-76 (pY128; top) and PLCγ1 (pY783; bottom) phosphorylation in J14-WT (•) versus J14-S376A cells (□) after normalization for the amount of 14-3-3ζ in each sample.

Article Snippet: The following mAbs were used: anti-FLAG M2 and anti-GST (Sigma-Aldrich); anti–SLP-76 (SLP-76/03) and anti–human CD3 (UCHT1; Serotec); anti-HA (F7; Santa Cruz Biotechnology, Inc.); anti-phosphorylated SLP-76 Y113 and Y128 (BD Biosciences); anti-CD28 (CD28.2; Immunotech); and anti–mouse CD3 (145-2C11; reference 40).

Techniques: Expressing, Mutagenesis, Western Blot, Construct

Journal: The Journal of Experimental Medicine

Article Title: A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

doi: 10.1084/jem.20062066

Figure Lengend Snippet: HPK-1 phosphorylates SLP-76 at serine 376. (A) Knockdown of HPK-1 expression in Jurkat T cells was performed by transient transfection of synthetic siRNAs. Cells were electroporated in the presence of medium alone (mock), control siRNAs (ctrl), or HPK-1–specific siRNAs (HPK1). After 72 h, cells were left unstimulated or activated by anti-CD3 mAb, and lysates were fractionated on Nu-PAGE gels. HPK-1 expression was assessed by anti–HPK-1 immunoblotting (top row). Phosphorylation of SLP-76 and PLC-γ1 was then assessed by immunoblotting with anti-pS376 (second row from top), anti-pY128 (third row from top), and anti–PLC-γ1 pY783 (fourth row from top). Equal protein loading in each lane was demonstrated by anti–SLP-76 and anti–14-3-3ζ immunoblotting (two bottom rows). (B) J14-WT cells were transiently transfected with either control siRNAs (ctrl) or HPK-1–specific siRNAs (HPK1) as in A. After 72 h, cells were left unstimulated or activated by anti-CD3 mAb for 10 min, and lysates were immunoprecipitated by anti-FLAG antibodies. Aliquots of the lysate were analyzed by immunoblotting with anti–HPK-1 or anti–SLP-76 antibodies (first and second row from top, respectively). Immunoprecipitates were immunoblotted with anti–SLP-76 (third row), anti-pS376 (fourth row), and anti–14-3-3ζ (bottom row). Quantification of band intensities and normalization by the amount of SLP-76 in each lane indicated that HPK-1 expression, pS376 phosphorylation, and 14-3-3ζ binding to SLP-76 were reduced in this experiment by 68, 58, and 50%, respectively. (C) COS-7 cells were transfected with FLAG–SLP-76 or HPK-1–HA constructs, WT, or a kinase-defective mutant (KD). After 36 h, cells were harvested, lysed, and immunoprecipitated with anti-FLAG (FLAG–SLP-76 transfection) or anti-HA antibodies (HPK-1 WT– or KD-transfected cells). FLAG–SLP-76 was eluted and mixed with bead-bound HPK-1–WT or –KD. Samples were then incubated for 30 min at 37°C in the presence or absence of ATP. Protein mixtures were analyzed by immunoblotting with anti-FLAG (top row), anti-HA (middle row), and anti-pS376 antibodies (bottom row).

Article Snippet: The following mAbs were used: anti-FLAG M2 and anti-GST (Sigma-Aldrich); anti–SLP-76 (SLP-76/03) and anti–human CD3 (UCHT1; Serotec); anti-HA (F7; Santa Cruz Biotechnology, Inc.); anti-phosphorylated SLP-76 Y113 and Y128 (BD Biosciences); anti-CD28 (CD28.2; Immunotech); and anti–mouse CD3 (145-2C11; reference 40).

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Binding Assay, Construct, Mutagenesis, Incubation