Journal: Frontiers in Immunology

Article Title: High-CBD Extract (CBD-X) Downregulates Cytokine Storm Systemically and Locally in Inflamed Lungs

doi: 10.3389/fimmu.2022.875546

Figure Lengend Snippet: CBD-X extract modulates TCR- signaling in human derived CD4 + T cells. CD4 + T cells were isolated from healthy donors by negative magnetic selection with the EasySep Direct Human CD4 + T cells Isolation Kit. Isolated CD4 + T cells were activated with 5 µg/ml anti-CD3 and 5 µg/ml anti-CD28 and treated with 1 or 2 µg/ml CBD-X extract or DMSO as a control. After an hour, the cells were collected and lysed with 100 µl 2x SB+βME. (A) Cell lysates were analyzed for the presence of phosphorylated STAT5, Lck and Zap70 by western blot analysis using pY694-STAT5, total STAT5, pY394-Lck, total Lck, pY319-Zap70 and total Zap70 antibodies. (B) Induction of phosphorylation was quantified relative to the specific total protein expression. Error bars represent the standard deviation of the means of three different donors, and they are expressed as average ± standard deviation (SD). SD were calculated in ratio to activated CD4 (treated with Anti-CD3/CD28), and data were analyzed by one-way ANOVA (Fisher’s LSD test with values p < 0.05 considered statistically significant, (*p <0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Mouse anti-phospho STAT5 and rabbit anti STAT5, rabbit anti-phospho Lck and mouse anti Lck, rabbit anti-phospho Zap70 and mouse anti–Zap70 as well as mouse anti β-Actin were obtained from Cell Signaling Technology (MA, USA).

Techniques: Derivative Assay, Isolation, Selection, Control, Western Blot, Phospho-proteomics, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: First Demonstration of Antigen Induced Cytokine Expression by CD4-1 + Lymphocytes in a Poikilotherm: Studies in Zebrafish ( Danio rerio )

doi: 10.1371/journal.pone.0126378

Figure Lengend Snippet: (A) Immunofluorescence staining of the zfCD4-1 molecule on zebrafish leukocytes. The cells stained with DAPI (blue) for counterstaining nuclei (left panel), the zfCD4-1 polyclonal followed by FITC labelled (green) secondary antibody (middle panel) and merged image (right panel) are shown. Scale bar = 10 μm). The arrow shows a cell expressing CD4 on its surface. (B) Cells from (A) were also visualized using a confocal microscope. (C) Double immunofluorescence staining of zebrafish peripheral lymphocytes in gut sections incubated with rabbit anti zfCD4-1 and mouse anti human ZAP70. ZAP70 + cells are green and CD4 + cells are red. Co-localization was confirmed by a Z-stack image analysis using Zeiss confocal microscopy. The data are representative results obtained from three independent experiments. (D) CD4-1 + cells present within the cuff of leukocytes surrounding the granulomas developed after M . marinum EspG5::Tn mutant infection of zebrafish 4 weeks earlier. Top images 200x, bottom images 400x. Left hand images used anti-CD4-1 serum, right hand images are controls.

Article Snippet: A rabbit monoclonal anti-human ZAP70 (BD 99F2, BD science) was purchased and used for double staining with the polyclonal anti-zfCD4-1 serum in immunohistochemistry.

Techniques: Immunofluorescence, Staining, Expressing, Microscopy, Double Immunofluorescence Staining, Incubation, Confocal Microscopy, Mutagenesis, Infection

Journal: Cell reports

Article Title: Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch

doi: 10.1016/j.celrep.2021.109280

Figure Lengend Snippet: (A) Schematic of TCR signaling and how optogenetic approaches can plug in at the step of Zap70:LAT clustering; see . (B) Design of the optogenetic constructs to compare dimerization and clustering of Zap70 and LAT. tRFP, TagRFP. (C) Schematic of protein complexes formed by light stimulation for the optogenetic constructs in (B). (D) tRFP-SSPB-Zap70 localization in NIH 3T3 cells. Images were taken prior to blue light illumination (gray border) or 5 min after illumination (blue border). Scale bars, 20 μm. (E) Quantification of cytosolic tRFP-SSPB-Zap70 fluorescence after illumination in both iLID-Only and iLID-Drop cells. (F) Quantification in change of the coefficient of variation (CV) of tRFP intensity for images taken in the membrane plane during 15 min of blue light illumination. For (E) and (F), envelope shows mean + SEM for at least 20 cells in each condition. See also and and .

Article Snippet: Total Zap70 mouse antibody , Cell Signaling Technologies , Cat # 2709S.

Techniques: Construct, Fluorescence

Journal: Cell reports

Article Title: Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch

doi: 10.1016/j.celrep.2021.109280

Figure Lengend Snippet: (A) Schematic of the Zap70:LAT interaction. Zap70 is phosphorylated on Tyr319 and then phosphorylates LAT rapidly at Tyr171, 191, and 226 and slowly at Tyr132. (B and C) Western blots and quantification of phospho-LAT Y132 and Y191 (B) and pY319-Zap70 (C) in the dark and after 20 min of blue light stimulation. (D) Schematic showing predicted responses to optogenetic clustering of LAT and Zap70 versus dimerization between LAT and Zap70 K362E , where the Zap70 mutation could potentially enable LAT phosphorylation from dimers alone. (E) Cartoon of iLID-Only Zap70 K362E. (F) Quantification of LAT pY191 and pY132 in cells expressing iLID-Only Zap70 K362E. (G) Quantification of the integrated signaling response from ErkKTR-irFP (C/N ratio) and GCaMP (fold-change). Boxes represent 25th–75th percentile, center line shows the mean, and whiskers show minimum and maximum values. n ≥ 30 data points are shown from three different experiments. Statistical comparisons were performed using Student’s t test across all independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also and .

Article Snippet: Total Zap70 mouse antibody , Cell Signaling Technologies , Cat # 2709S.

Techniques: Western Blot, Mutagenesis, Expressing

Journal: Cell reports

Article Title: Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch

doi: 10.1016/j.celrep.2021.109280

Figure Lengend Snippet: (A) Images of kinase-dead tRFP-Zap70 K369R (Zap70 KD ) in iLID-Drop cells. Images show cytosolic and membrane planes. Scale bars, 20 μm. (B) Western blot and quantification of pY319-Zap70 for the indicated cell lines. (C) Images of tRFP-Zap70 localization in LAT FFF (LAT Y171F, Y191F, Y226F) iLID-Drop cells. (D) Western blot and quantification of pY319-Zap70 in the indicated cell lines. (E) Schematic of positive feedback loop between LAT and Zap70 in the iLID-Drop clusters. No increase in pZap70 is observed in Zap70 KD or LAT FFF cells, demonstrating that this upstream event depends on downstream steps. For (A)–(D), cells were incubated in the dark (gray) or after 20 min of light (blue). Graphs display mean ± SEM for all independent biological replicates (points). All statistical comparisons were performed using Student’s t test using all independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also .

Article Snippet: Total Zap70 mouse antibody , Cell Signaling Technologies , Cat # 2709S.

Techniques: Western Blot, Incubation

Journal: Cell reports

Article Title: Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch

doi: 10.1016/j.celrep.2021.109280

Figure Lengend Snippet: (A) Schematic SFK inhibitor treatment in iLID-Drop cells. (B) Western blot and quantification of phospho-Zap70 after 20-min treatment with PP1 and PP2 versus DMSO. (C) Schematic of experiments in iLID-Drop SYF cells. (D) Western blot and quantification of pY319-Zap70 in indicated cell lines. (E and F) AUC of ErkKTR-irFP (E) or GCaMP (F) responses for iLID-Drop SYF cells. Boxes represent 25th–75th percentile, center line shows the mean, and whiskers show minimum and maximum values. n ≥ 20 cells from two different experiments. (G) Schematic of potential SFK inputs into the LAT-Zap70 feedback circuit. (H) Schematic of experiments in Src ΔSH2−3 iLID-Drop SYF cells. (I) Western blot and quantification of pY319-Zap70 in the indicated SYF cell lines. Bar graphs display mean ± SEM and independent biological replicates (points). All statistical comparisons were performed using Student’s t test using all independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also .

Article Snippet: Total Zap70 mouse antibody , Cell Signaling Technologies , Cat # 2709S.

Techniques: Western Blot

Journal: Cell reports

Article Title: Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch

doi: 10.1016/j.celrep.2021.109280

Figure Lengend Snippet: (A) Schematic of the model. In the dark, the modeled cell consists of two well-mixed compartments (indicated by the stir propellors) representing the cytosol and the membrane. In iLID-Only simulations, light stimulation recruits Zap70 to the membrane, while in in iLID-Drop, this effect is combined with a 10-fold drop in the membrane compartment volume. (B) Simulated cellular concentrations of pZap70 (yellow) and pLAT (brown) following light stimulation (blue) in six different experimental scenarios. (C and D) The modeled ratio of pLAT to total LAT is shown as a function of LAT’s partition coefficient on the membrane (C) and the cellular concentration of Src (D). Curves represent iLID-Drop simulations in dark (gray) and light (blue) conditions. (E) iLID-Drop simulations (lines) and experimental replicates (points) for the ratio of pLAT to total LAT in the presence of the Src inhibitor PP2 in SYF iLID-Drop fibroblasts expressing either wt Src (blue) or Src ΔSH2−3 (purple). See also and – .

Article Snippet: Total Zap70 mouse antibody , Cell Signaling Technologies , Cat # 2709S.

Techniques: Concentration Assay, Expressing

Journal: Cell reports

Article Title: Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch

doi: 10.1016/j.celrep.2021.109280

Figure Lengend Snippet: (A) Schematic of central hypothesis: LAT clustering triggers positive feedback, leading to a T cell signaling response. (B) LAT-deficient JCAM2.5 Jurkat T cells were transduced with the GCaMP biosensor and either LAT-Cry2 or a light-insensitive variant (LAT-Cry2 BLI ). (C) Representative images for JCaM2.5 GCaMP cells transduced with nothing, LAT-Cry2BLI, or LAT-Cry2. Dark (top), illuminated (bottom), and anti-CD3 antibody (red) stimulation conditions are shown. (D) Quantification of (C) showing the fraction of cells exhibiting a sustained increase in GCaMP fluorescence after stimulation. Each data point represents at least 50 cells from independent experiments. Boxes represent median ± quartiles. **p < 0.01 and ***p < 0.001 as derived from Student’s t test. (E) Schematic of cluster-localized positive feedback among LAT, SFKs, and Zap70. A low amount of basally phosphorylated Zap70 performs some leaky phosphorylation of LAT. Phosphorylated LAT then recruits and activates SFKs through SH2-pTyr and SH3-proline rich motif interactions. Finally, active SFKs phosphorylate and activate additional Zap70 molecules within the cluster, completing the feedback loop. See also and .

Article Snippet: Total Zap70 mouse antibody , Cell Signaling Technologies , Cat # 2709S.

Techniques: Transduction, Variant Assay, Fluorescence, Derivative Assay

Journal: Cell reports

Article Title: Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch

doi: 10.1016/j.celrep.2021.109280

Figure Lengend Snippet:

Article Snippet: Total Zap70 mouse antibody , Cell Signaling Technologies , Cat # 2709S.

Techniques: Recombinant, Clone Assay, Plasmid Preparation, Software

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet:

Article Snippet: Finally, cells were pooled and stained with anti-CD3 BV510 (Biolegend, #300448), anti-CD4 PE (Biolegend, #357404), anti-CD8 AF700 (Biolegend, #300920), anti-pSTAT1-Y701 AF647 (Cell Signaling, #8009S), anti-pSTAT1-S727 AF488 (Biolegend, #686410), anti-pSTAT3-Y705 AF488 (Biolegend, #651006) and anti-pSTAT3-S727 AF647 (Biolegend, #698913), anti-pSTAT4-Y693 AF488 (BD Biosciences, #558136), anti-pSTAT5-Y694 AF647 (Cell Signaling, #9365S), anti-pSTAT6-Y641 AF488 (BD Biosciences, #612600), anti-pERK-T202/Y204 AF488 (eBiosciences, #53-9109-41), anti-pAKT-S473 AF488 (Cell Signaling, #4071S), anti-pAKT-T308 AF647 (Cell Signaling, #48646S), anti-pP90RSK-S380 AF488 (Cell Signaling, #13588S), anti-pS6R-S240/S244 AF488 (Cell Signaling, #5018S), anti-pS6R-S235/S236 AF647 (Cell Signaling, #4851S), anti-pZAP70-Y319/pSYK-Y352 AF647 (Cell Signaling, #82975S), anti-pCREB-S133 AF488 (Cell Signaling, #9187S), anti-pHIS3-S10 AF647 (Cell Signaling, #9716S), anti-pGSK3β-S9 AF647 (Cell Signaling, #14332S),anti-pCFOS-S32 AF647 (Cell Signaling, #8677S), anti-IRF1 AF647 (Cell Signaling, #14105S), anti-IRF4 AF647 (Biolegend, #646408), anti-IRF7 AF647 (Biolegend, #656007), anti-GATA3 AF488 (Biolegend, #653807), anti-TBET AF647 (Biolegend, #644803), anti-HIF1α AF488 (Biolegend, #359707), anti-MYC AF488 (Cell Signaling, #12855S), anti-O-GlcNAC AF647 (NOVUS Biologicals, #NB300-524AF647), anti-STAT3 APC (BD Biosciences, #560392) and anti-PLCγ1 AF647 (BD Biosciences, #557883).

Techniques: Purification, Recombinant, Phospho-proteomics, Software

Journal: Science signaling

Article Title: Superresolution imaging of the cytoplasmic phosphatase PTPN22 links integrin-mediated T cell adhesion with autoimmunity.

doi: 10.1126/scisignal.aaf2195

Figure Lengend Snippet: Fig. 7. Mechanistic model for the regulation of integrin signaling by PTPN22. The transition of LFA-1 from a low- or intermediate-affinity state (middle) to a high- affinity state (left) is characterized by the phosphorylation of Lck, ZAP70, and Vav, which are associated with the cytoplasmic tail of the b2 subunit of LFA-1. Subse- quently, spatiotemporal regulation of LFA-1 signals is mediated by the dispersal of PTPN22 and Csk from clusters, the disassociation of PTPN22 and Csk from PAG at the plasma membrane, and the increased association of PTPN22 with Csk through the P1 domain of PTPN22 and the SH3 domain of Csk. PTPN22-Csk complexes target their phosphorylated substrates in the LFA-1 signaling complex, which leads to the attenuation of LFA-1 signaling. Although the declustering of the loss-of-function PTPN22-W620 mutant is preserved, the binding of PTPN22-W620 to Csk is impaired (right). The mutant phosphatase is not retained at the plasma membrane, and in the absence of membrane-proximal binding partners, such as Csk, PTPN22-W620 diffuses away from the plasma membrane. As a consequence, LFA-1 signal in- tensity is augmented and sustained, further promoting LFA-1 clustering at the cell surface and increasing integrin-dependent adhesion (right). The spatiotemporal organization of PTPN22-R620 (PTP), PTPN22-W620 (PTP-W), Csk, talin (Tal), Lck, Vav1 (Vav), ZAP70 (Zap), and kindlin is illustrated. P denotes phosphorylation on tyrosine residues.

Article Snippet: Antibodies specific for Vav1 (C-14), ZAP70 (1E7.2), Lck (3A5), and Csk (C20) were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Clinical Proteomics, Membrane, Mutagenesis, Binding Assay

Journal: Frontiers in Immunology

Article Title: CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

doi: 10.3389/fimmu.2018.03115

Figure Lengend Snippet: CCR7 triggering recruits and activates ZAP70. (A) Micrograph and quantification (box and whisker plot, n > 50 cells) of CCR7-ZAP70 interaction in human PBLs stimulated or not with 0.5 μg/ml CCL19 or CCL21 for 15 min assessed by PLA. Representative epifluorescence images derived from one donor out of three; scale bar, 10 μm. (B) Confocal images of CCR7-YFP2:ZAP70-YFP1 interaction determined by BiFC in transfected HEK293 cells stimulated or not with 0.5 μg/ml CCL19 or CCL21 for 1 h. One experiment out of three; scale bars, 25 μm. (C) Quantification of CCR7-YFP2:ZAP70-YFP1 interaction by BiFC assessed by flow cytometry after 1 h stimulation with 0.5 μg/ml CCL19 or CCL21. Mean ± SEM of five independent experiments. (D) Confocal images and quantification (Pearson's coefficient, n > 15 cells) of the co-localization between CCR7-YFP and ZAP70 in 3B11 T cells stimulated or not with 0.5 μg/ml CCL21 for 1 h either alone or conjugated with HEL peptide-loaded LK35.2 APCs. Representative images derived from one of three independent experiments; scale bar, 5 μm. (E) Quantification of ZAP70 activation determined by FRET efficiency of the ROZA-XL FRET biosensor in Jurkat T cells upon CCR7 and/or TCR stimulation [5 μg/ml anti-CD3 (clone OKT3)]. Mean ± SEM of 10 cells per condition derived from one out of three independent experiments. ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns, not significant.

Article Snippet: The following antibodies were used: PE-labeled anti CD69 (clone FN50) (Bio-Rad, Hercules, CA, USA), PE-labeled anti-IL-2 (BD Pharmigen, Franklin Lakes, NJ, USA), anti-human CCR7 used for immune fluorescence (SAB4500329) (Sigma-Aldrich, St. Louis, MO, USA), anti-human CCR7 (LifeSpan Biosciences, Seattle, WA, USA) used for PLA, anti-human Vav1 (9C1) (Abnova, Taipei City, Taiwan) used for PLA, anti-human CCR7 APC (FAB197A) (R&D Systems, Minneapolis, MN, USA), anti-humanCD3ζ (ab188850) (abcam, Cambridge, United Kingdom), anti-CD3 (ab5690) (abcam), anti-CD3 (clone OKT3; Janssen-Cilag, Beerse, Belgium), anti-ZAP70 (D1C10E) XP® Rabbit mAb (Cell Signaling, Danvers, MA), anti-ZAP70 (phospho Y319) antibody (ab131270) (abcam), anti-YFP1 (E385) (abcam), anti-YFP2 (11814460001) (Roche, Basel, Switzerland), monoclonal anti-HA-HRP (clone HA7) (Sigma-Aldrich), anti-HA antibody (clone HA7) (Sigma-Aldrich), anti-CD11a antibody [EP1285Y] (ab52895) (abcam), anti-CD18 antibody [MEM-48] (ab657) (abcam), mAb24 anti-CD11+CD18 antibody [24] (ab13219) (abcam).

Techniques: Whisker Assay, Derivative Assay, Transfection, Flow Cytometry, Activation Assay

Journal: Frontiers in Immunology

Article Title: CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

doi: 10.3389/fimmu.2018.03115

Figure Lengend Snippet: Chemokine-driven ZAP70 recruitment to CCR7 depends on Src. (A) Confocal images of CCR7-YFP2:ZAP70-YFP1 BiFC upon chemokine stimulation (0.5 μg/ml for 1 h) in HEK293 cells that were left untreated or pre-treated with PP2 (10 μM), or co-transfected with a kinase-dead variant of Src (SrcKD). Representative images derived from one out of three independent experiments; scale bars, 25 μm. (B,C) Quantification of CCR7-YFP2:ZAP70-YFP1 BiFC assessed by flow cytometry in transfected HEK293 cells pre-treated or not with PP2 (10 μM) (B) or co-transfected with SrcKD (C) . Mean ± SEM of three (C) or four (B) independent experiments. Cells were stimulated with 0.5 μg/ml CCL19 or CCL21 for 1 h. (D,E) Quantification of CCR7-YFP2:ZAP70-YFP1 (D) and CCR7-Y155F-YFP2:ZAP70-YFP1 (E) interaction determined by BiFC and flow cytometry in transfected HEK293 cells that were stimulated or not with 0.5 μg/ml CCL19 or CCL21 for 1 h. Mean ± SEM of three independent experiments. (F) Quantification of CCR7-YFP2:ZAP70-YFP1 BiFC in HEK293 cells treated or not with PTx (100 ng/ml) in presence or absence of 0.5 μg/ml CCL19 or CCL21 for 1 h. Mean ± SEM of three independent experiments. (G) Quantification of CCR7-DNY-YFP2:ZAP70-YFP1 assessed by BiFC and flow cytometry in transfected HEK293 cells. Mean ± SEM of three independent experiments. (H) Quantification of CCR7 WT, CCR7-V317I, or CCR7-A315G fused to YFP2 interaction with ZAP70-YFP1 determined by BiFC and flow cytometry in transfected HEK293 cells. Cells were stimulated or not with 0.5 μg/ml CCL19 or CCL21 for 1 h. Mean ± SEM of four independent experiments. (I) Quantification of CD69 surface expression on human PBLs, pretreated with PTx (100 ng/ml) or PP2 (10 μM) or left untreated, upon TCR stimulation using anti-CD3/CD28 coated beads and/or CCR7 stimulation (0.5 μg/ml CCL19 or CCL21) for 20 h measured by flow cytometry. Mean ± SEM of four individual donors. * p < 0.1; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns, not significant.

Article Snippet: The following antibodies were used: PE-labeled anti CD69 (clone FN50) (Bio-Rad, Hercules, CA, USA), PE-labeled anti-IL-2 (BD Pharmigen, Franklin Lakes, NJ, USA), anti-human CCR7 used for immune fluorescence (SAB4500329) (Sigma-Aldrich, St. Louis, MO, USA), anti-human CCR7 (LifeSpan Biosciences, Seattle, WA, USA) used for PLA, anti-human Vav1 (9C1) (Abnova, Taipei City, Taiwan) used for PLA, anti-human CCR7 APC (FAB197A) (R&D Systems, Minneapolis, MN, USA), anti-humanCD3ζ (ab188850) (abcam, Cambridge, United Kingdom), anti-CD3 (ab5690) (abcam), anti-CD3 (clone OKT3; Janssen-Cilag, Beerse, Belgium), anti-ZAP70 (D1C10E) XP® Rabbit mAb (Cell Signaling, Danvers, MA), anti-ZAP70 (phospho Y319) antibody (ab131270) (abcam), anti-YFP1 (E385) (abcam), anti-YFP2 (11814460001) (Roche, Basel, Switzerland), monoclonal anti-HA-HRP (clone HA7) (Sigma-Aldrich), anti-HA antibody (clone HA7) (Sigma-Aldrich), anti-CD11a antibody [EP1285Y] (ab52895) (abcam), anti-CD18 antibody [MEM-48] (ab657) (abcam), mAb24 anti-CD11+CD18 antibody [24] (ab13219) (abcam).

Techniques: Transfection, Variant Assay, Derivative Assay, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

doi: 10.3389/fimmu.2018.03115

Figure Lengend Snippet: Vav1 recruitment to ZAP70 upon CCR7 stimulation depends on Src kinase and Y315-phosphorylated ZAP70. (A) Micrograph and quantification (box and whisker plot, n > 35 cells) of Vav1-ZAP70 interaction in primary human PBLs assessed by PLA. Cells were stimulated or not with 0.5 μg/ml CCL19/CCL21 for 15 min. Representative epifluorescence images derived from one donor out of three; scale bar, 10 μm. (B) Confocal images of ZAP70-YFP1:Vav1-YFP2 interaction determined by BiFC in HEK293 cells expressing CCR7-HA upon stimulation with CCL19 or CCL21 (0.5 μg/ml, 1 h). Representative images derived from one out of three independent experiments; scale bar, 25 μm (C) Quantification of ZAP70-YFP1:Vav1-YFP2 interaction determined by BiFC and flow cytometry in HEK293 cells expressing CCR7-HA stimulated with CCL19 or CCL21 (0.5 μg/ml, 1 h). Mean ± SEM of five independent experiments. (D) Quantification of ZAP70-Y315F-YFP1:Vav1-YFP2 interaction determined by BiFC and flow cytometry in HEK293 cells expressing CCR7-HA stimulated with CCL19 or CCL21 (0.5 μg/ml, 1 h). Mean ± SEM of three independent experiments. (E,F) Quantification of ZAP70-YFP1:Vav1-YFP2 interaction assessed by BiFC and flow cytometry in HEK293 cells expressing CCR7-HA after PP2 (10 μM) treatment (E) or SrcKD co-transfection (F) . Mean ± SEM of three (F) or four (E) independent experiments. *** p < 0.001; **** p < 0.0001. ns, not significant.

Article Snippet: The following antibodies were used: PE-labeled anti CD69 (clone FN50) (Bio-Rad, Hercules, CA, USA), PE-labeled anti-IL-2 (BD Pharmigen, Franklin Lakes, NJ, USA), anti-human CCR7 used for immune fluorescence (SAB4500329) (Sigma-Aldrich, St. Louis, MO, USA), anti-human CCR7 (LifeSpan Biosciences, Seattle, WA, USA) used for PLA, anti-human Vav1 (9C1) (Abnova, Taipei City, Taiwan) used for PLA, anti-human CCR7 APC (FAB197A) (R&D Systems, Minneapolis, MN, USA), anti-humanCD3ζ (ab188850) (abcam, Cambridge, United Kingdom), anti-CD3 (ab5690) (abcam), anti-CD3 (clone OKT3; Janssen-Cilag, Beerse, Belgium), anti-ZAP70 (D1C10E) XP® Rabbit mAb (Cell Signaling, Danvers, MA), anti-ZAP70 (phospho Y319) antibody (ab131270) (abcam), anti-YFP1 (E385) (abcam), anti-YFP2 (11814460001) (Roche, Basel, Switzerland), monoclonal anti-HA-HRP (clone HA7) (Sigma-Aldrich), anti-HA antibody (clone HA7) (Sigma-Aldrich), anti-CD11a antibody [EP1285Y] (ab52895) (abcam), anti-CD18 antibody [MEM-48] (ab657) (abcam), mAb24 anti-CD11+CD18 antibody [24] (ab13219) (abcam).

Techniques: Whisker Assay, Derivative Assay, Expressing, Flow Cytometry, Cotransfection

Journal: Frontiers in Immunology

Article Title: CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

doi: 10.3389/fimmu.2018.03115

Figure Lengend Snippet: ZAP70 controls CCR7-mediated inside-out signaling to LFA-1. (A) Arrest of Jurkat P116 cells lacking ZAP70 and Jurkat P116 cells reconstituted with ZAP70-GFP on immobilized ICAM-1 in the presence or absence of CCL19, CCL21, or MgCl 2 . Mean ± SEM of three independent experiments. (B) Binding of the β 2 -integrin high-affinity reporter mAb24 to Jurkat P116 and Jurkat P116 ZAP70-GFP cells upon CCR7 triggering for 10 min. Mean ± SEM of five independent experiments. (C–E) Confocal images of CD11a staining on Jurkat P116, Jurkat P116 ZAP70-GFP, or Jurkat P116 ZAP70-K369R-GFP cells before and after chemokine stimulation (0.5 μg/ml, 10 min). (C) no pre-treatment. (D) pre-treatment with DMSO or piceatannol. Representative images derived from one out of three independent experiments; scale bar, 10 μm. **** p < 0.0001. ns, not significant.

Article Snippet: The following antibodies were used: PE-labeled anti CD69 (clone FN50) (Bio-Rad, Hercules, CA, USA), PE-labeled anti-IL-2 (BD Pharmigen, Franklin Lakes, NJ, USA), anti-human CCR7 used for immune fluorescence (SAB4500329) (Sigma-Aldrich, St. Louis, MO, USA), anti-human CCR7 (LifeSpan Biosciences, Seattle, WA, USA) used for PLA, anti-human Vav1 (9C1) (Abnova, Taipei City, Taiwan) used for PLA, anti-human CCR7 APC (FAB197A) (R&D Systems, Minneapolis, MN, USA), anti-humanCD3ζ (ab188850) (abcam, Cambridge, United Kingdom), anti-CD3 (ab5690) (abcam), anti-CD3 (clone OKT3; Janssen-Cilag, Beerse, Belgium), anti-ZAP70 (D1C10E) XP® Rabbit mAb (Cell Signaling, Danvers, MA), anti-ZAP70 (phospho Y319) antibody (ab131270) (abcam), anti-YFP1 (E385) (abcam), anti-YFP2 (11814460001) (Roche, Basel, Switzerland), monoclonal anti-HA-HRP (clone HA7) (Sigma-Aldrich), anti-HA antibody (clone HA7) (Sigma-Aldrich), anti-CD11a antibody [EP1285Y] (ab52895) (abcam), anti-CD18 antibody [MEM-48] (ab657) (abcam), mAb24 anti-CD11+CD18 antibody [24] (ab13219) (abcam).

Techniques: Binding Assay, Staining, Derivative Assay

Journal: Osong Public Health and Research Perspectives

Article Title: Ingenol Protects Human T Cells From HIV-1 Infection

doi: 10.1016/j.phrp.2011.07.001

Figure Lengend Snippet: Western blot analysis of iNOs and ZAP-70 in MT4 cells. Ingenol treatment during HIV infection resulted in recovery of iNOS and ZAP-70. MT-4 cells were infected with HIV-1 IIIB at multiplicity of infection (MOI; 0.004 [50 TCID 50 ]) after 5 μM ingenol treatment then protein expression in cell lysate was analyzed by western blot 24 hours after HIV infection. iNOS, inducible nitric oxide synthase; ZAP-70, zeta chain-associated protein kinase 70. β-actin was used as loading control.

Article Snippet: After transfer of separated proteins to Immobilon™–P PVDF membrane (Millipore, Danvers, MA), primary antibodies were applied for hybridization: rabbit anti-human inducible nitric oxide synthase (iNOS) antibody (Abcam, Cambridge, MA) and rabbit anti-human zeta chain-associated protein kinase 70 (ZAP-70) antibody (Cell Signaling, Danvers, MA).

Techniques: Western Blot, Infection, Expressing