Journal:

Article Title: The mutant leucine-zipper domain impairs both dimerization and suppressive function of Foxp3 in T cells

doi: 10.1073/pnas.0600225103

Figure Lengend Snippet: The glutamic acid residue in the leucine-zipper domain of Foxp3 is required for homodimerization. (A) FLAG- and Myc-tagged Foxp3 or Foxp3ΔE were cotransfected for 48 h. After harvesting of cells, lysates were incubated with FLAG antibody (M2)-conjugated agarose beads, washed and boiled with sample buffer for SDS/PAGE. Immunoprecipitates were analyzed by anti-Myc Ab. Lysates were also analyzed for protein expression by using anti-FLAG and anti-Myc Abs. RLU, relative luciferase unit. (B–D) Each null vector, Foxp3, and Foxp3 ΔE was cotransfected with the FKH luciferase construct in Jurkat T cells with lipid-mediated transfection (B). After 48 h, luciferase activity was measured (C and D). Each NFAT and NF-κB luciferase construct was cotransfected with null vector, Foxp3, or Foxp3ΔE in HEK293 cells. After 48-h transfection, cells were harvested and assessed for luciferase activity. All samples were normalized by cotransfection and analysis of TK-Renilla activity. A representative result from at least three independent experiments is shown.

Article Snippet: The Jurkat Tet-on cell line was purchased from Clontech (BD Biosciences) and infected with the pREV-TRE2 Foxp3 expression vector.

Techniques: Residue, Incubation, SDS Page, Expressing, Luciferase, Plasmid Preparation, Construct, Transfection, Activity Assay, Cotransfection

Journal: Journal of Translational Medicine

Article Title: AQP5-1364A/C polymorphism and the AQP5 expression influence sepsis survival and immune cell migration: a prospective laboratory and patient study

doi: 10.1186/s12967-016-1079-2

Figure Lengend Snippet: Western blot and migration assay of transfected Jurkat-cells and AQP5-immunostaining of neutrophils obtained from healthy donors a Western blot from whole cell lysates of Jurkat-cells ( lane u untransfected cells, lane c transfected with control plasmid, lanes 1 – 3 AQP5_pReceiver transfected Jurkat cells) with AQP5 and ACTB antibody; As indicated by the stronger band in lanes 1 – 3 , Jurkat cells could successfully be stable transfected; b Migration assay performed with cell culture inserts utilizing transfected Jurkat cells stimulated with SDF-1α and counted after 22 h (n = 6, 6); AQP5 overexpressing (AQP5_pRec) cells showed increased migration (p = 0.009) compared to control cells (pRec). c Representative examples of immunostaining with AQP5 antibody of neutrophils obtained from two healthy AA genotype carriers showing a considerable amount of AQP5 protein ( brown precipitate ). In neutrophils of two CC genotype carriers no clear AQP5 protein expression is detected. Every staining was performed in duplicate and eight random photographs were taken from each slide, 40× magnification

Article Snippet: Stable transfection with Jurkat T-cells was performed using an AQP5_pReceiver EX-T1015-M09 vector (Human Full ORF-Clone, NM-001651, pReceiver-M09) or a pReceiverM09 (GeneCopeia, Rockville, MD, USA).

Techniques: Western Blot, Migration, Transfection, Immunostaining, Control, Plasmid Preparation, Cell Culture, Expressing, Staining

Journal: Function

Article Title: Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry

doi: 10.1093/function/zqac033

Figure Lengend Snippet: Effect of cannabinoids on NFAT activity in standard culture medium. Pure cannabinoids were tested at 5 µ m against NFAT activity. Jurkat T cells expressing NFAT reporter gene were pretreated with the cannabinoids before stimulation using 1 µ m ionomycin and 20 ng/mL PMA. Bioluminescence emitted by NFAT-mediated luciferase expression was measured in arbitrary units 5 h poststimulation. All data points represent mean ± SEM of 3 independent runs ( N = 3), with each run including n = 3 replicates per condition. * P < .05, ** P < .01. Key: NT = nontreated cells, NS = nonstimulated, MeOH = methanol, MeCN = acetonitrile, DMSO = dimethylsulfoxide.

Article Snippet: Jurkat NFAT reporter cell line (BPS Bioscience, San Diego, CA, USA) was maintained in RPMI 1640 medium supplemented with 10% FBS.

Techniques: Activity Assay, Expressing, Luciferase

Journal: Function

Article Title: Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry

doi: 10.1093/function/zqac033

Figure Lengend Snippet: CBGA inhibitory potency is decreased by increasing serum concentrations. Serum-dependent effects of CBGA on I CRAC and NFAT activity in the presence of 0%, 1%, and 10% FBS. (A) Time course of CRAC currents recorded in Jurkat T cell in whole-cell patch-clamp experiments. Bath solution was supplemented with 0%, 1%, or 10% FBS (0% serum data is the same as in ). To elicit the active calcium depletion from the ER, 50 µ m IP 3 in the recording pipette was utilized. The resulting CRAC currents were allowed to reach maximal activation prior to extracellular application of CBGA at various concentrations. Current amplitudes were measured at −120 mV for each voltage ramp and were normalized to the control at 120 s. n represents the number of cells patched for each condition. (B) Concentration–response effects of CBGA on NFAT activity in the presence of 0%, 1%, and 10% FBS. A 10- and 12-point concentration-response curves, ranging from 100 n m to 100 µ m and 100 n m to 300 µ m , respectively, were established. The 12-point d/r was used for 10% FBS condition due to the weak activity of CBGA in the ten-point concentration range under this condition. Values are average ± SEM of 12–15 replicates, normalized to the control (cells treated with PMA + Ionomycin only).

Article Snippet: Jurkat NFAT reporter cell line (BPS Bioscience, San Diego, CA, USA) was maintained in RPMI 1640 medium supplemented with 10% FBS.

Techniques: Activity Assay, Patch Clamp, Transferring, Activation Assay, Concentration Assay

Journal: Function

Article Title: Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry

doi: 10.1093/function/zqac033

Figure Lengend Snippet: Isobolographic analysis of CBGA and other cannabinoids against SOCE. (A) An isobole showing dose pairs of CBGA and CBD that are expected to inhibit SOCE by 50%. The y- and the x-intercepts represent the IC 50 values of CBGA and CBD, respectively. (B) Inhibition of SOCE observed when Jurkat NFAT cells were treated with concentration pairs of CBGA and CBD. The dashed line depicts the expected 50% inhibition of SOCE at each dose pair. The bold line connects the actual inhibition observed when treated separately with CBGA and the other cannabinoid (CBD) at individual IC 50 . The bars represent the observed inhibition when treated with various concentration pairs of the two compounds. Here, the bars fall on the bold line, indicating that the two compounds follow a simple additivity behavior. (C) Dose pairs of CBGA and THCVA show deviation from expected inhibition. The green bars above the bold line suggest supra-additive (synergistic) effects between the two compounds. (D) Concentration pairs of CBGA and Δ8-THC show deviation in the opposite direction, suggesting subadditive (inhibitory) effects.

Article Snippet: Jurkat NFAT reporter cell line (BPS Bioscience, San Diego, CA, USA) was maintained in RPMI 1640 medium supplemented with 10% FBS.

Techniques: Inhibition, Concentration Assay

Journal: Cancer immunology research

Article Title: KIR3DL3 is an inhibitory receptor for HHLA2 that mediates an alternative immunoinhibitory pathway to PD1

doi: 10.1158/2326-6066.CIR-20-0315

Figure Lengend Snippet: (A) Jurkat IL2-reporter T cells expressing KIR3DL3 were co-cultured with CHO cells expressing anti-CD3 scFV, CHO cells coexpressing anti-CD3 scFV and HHLA2, or untransfected CHO cells in the presence or absence of CD28 mAb as indicated. Luciferase activity represented as relative light units (RLU). (B, C) Jurkat IL2-reporter T cells expressing KIR3DL3 were co-cultured with CHO cells coexpressing anti-CD3 scFV and HHLA2 in the presence of CD28 mAb and (B) HHLA2 mAbs or (C) KIR3DL3 mAbs. Fold activation of IL2 reporter luciferase activity is presented as mean ± S.D. (n≥3; **** P≤0.0001).

Article Snippet: Jurkat cells (clone E6–1) expressing IL2_promoter Luc_pcDNA (BPS Bioscience; cat. #60481) were transfected with KIR3DL3 (pEF-Puro) by electroporation.

Techniques: Expressing, Cell Culture, Luciferase, Activity Assay, Activation Assay

Journal: Scientific Reports

Article Title: Ligand non-competitive GITR antibody prevents formation of the obligatory signal-triggering GITRL: GITR stoichiometry

doi: 10.1038/s41598-025-32541-6

Figure Lengend Snippet: Discovery and characterization of ligand non-competitive NFκB neutralizing anti-GITR antibody. ( A ) Discovery of ligand competitive and non-competitive antibodies from mice immunization campaign measured by antibody mediated % inhibition of human GITRL binding to recombinant human GITR protein via competition ELISA. ( B ) Recombinant GITRL protein induces NFκB signaling in GITR-NFκB-Luciferase-Jurkat cell line. ( C ) Bivalent ligand non-competitive anti-GITR antibody (hGITR-nc-Ab-#1) neutralizes recombinant GITRL protein induced NFκB signaling. ( D ) Monovalent one-armed ligand non-competitive anti-GITR antibody (hGITR-nc-Ab-#1-OA) also neutralizes recombinant GITRL protein induced NFκB signaling.

Article Snippet: GITRL-GITR mediated NFκB signaling was measured using GITR-NFκB-Luciferase-Jurkat cell line (BPS bioscience).

Techniques: Inhibition, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase

Journal: Cell Reports Medicine

Article Title: Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors

doi: 10.1016/j.xcrm.2021.100457

Figure Lengend Snippet: Investigation of co-stimulation delivered by pCAR-M/34 (A) Carboxyfluorescein N-succinimidyl ester (CFSE)-labeled M-CSFR-specific CAR and pCAR T cells were stimulated for 24 h on T47D or T47D FMS tumor monolayers and then flow sorted prior to RNA extraction. Gene set enrichment analysis (GSEA) demonstrated significant cytokine pathway enrichment in pCAR-M/34 T cells compared with all controls. (B) Enriched cytokine-signaling pathways (false discovery rate [FDR] < 0.25; p < 0.1) in pCAR-M/34 pCAR T cells. p < 0.05 for all listed pathways, unless indicated otherwise. (C) “Blue pink o’gram” heatmap of cytokine gene expression in pCAR-M/34 and control T cell populations following stimulation on T47D FMS tumor monolayers. (D) Engineered Jurkat NF-κB reporter cells were co-cultured with T47D or T47D FMS cells for 5 h. Cell lysates were then analyzed for luciferase activity (mean ± SD, n = 3). Effect of tumor necrosis factor alpha (TNF-α) is shown as positive control. M-CSFR-specific CAR and pCAR T cells were re-stimulated each week as described in Figure 2 C. See Figure S3 for additional data.

Article Snippet: NF-κB luciferase reporter Jurkat , BPS Bioscience (distributed by Tebu-Bio) , Cat# 60651.

Techniques: Labeling, RNA Extraction, Expressing, Cell Culture, Luciferase, Activity Assay, Positive Control

Journal: Cell Reports Medicine

Article Title: Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors

doi: 10.1016/j.xcrm.2021.100457

Figure Lengend Snippet:

Article Snippet: NF-κB luciferase reporter Jurkat , BPS Bioscience (distributed by Tebu-Bio) , Cat# 60651.

Techniques: Recombinant, Staining, Transfection, Enzyme-linked Immunosorbent Assay, Luciferase, Expressing, Clone Assay, Software