14-7049-85

antibody from Invitrogen Antibodies

Targeting: IL4 BCGF-1, BCGF1, BSF1, IL-4, MGC79402

Provider product page for 14-7049-85

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Antibody data

Antibody Data
Antigen structure
References [17]
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Validations
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Product number: 14-7049-85 - Provider product page
Provider: Invitrogen Antibodies
Product name: IL-4 Monoclonal Antibody (8D4-8), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The 8D4-8 antibody reacts with human interleukin-4 (IL-4), a 15-19 kDa cytokine secreted by Th2 cells. Applications Reported:The 8D4-8 antibody has been reported for use in capture of human IL-4 by ELISA and in intracellular staining for flow cytometric analysis. Applications Tested: The 8D4-8 antibody has been tested as the capture antibody in a sandwich ELISA for analysis of human Interleukin-4 (IL-4) in combination with the biotinylated MP4-25D2 (Product # 13-7048-81) antibody for detection and recombinant human IL-4 (Product # 14-8049-80) as the standard. A suitable range of concentrations of this antibody for ELISA capture is 1-4 µg/mL. A standard curve consisting of doubling dilutions of the recombinant standard over the range of 500 pg/mL - 4 pg/mL should be included in each ELISA plate. The fluorochrome-conjugated 8D4-8 antibody has been tested by intracellular staining for flow cytometric analysis. This can be used at less than or equal to 0.5 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation:Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: 8D4-8
Vial size: 500 µg
Concentration: 0.5 mg/mL
Storage: 4° C

IL4 protein structure - 14-7049-85 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. piR30840 regulates the development of Th2 lymphocytes. ( A and B ) Human CD4 naive cells were cultured in a conditioned medium for Th2 development and then transfected with an s30840 (A) or piR30840 inhibitor (B). IL-4 expression was detected by qRT-PCR and western blotting analyses. P < 0.01. The data represents three independent experiments. ( C ) Detection of IL-4 expression in human PBMCs isolated from humanized NOG mice. IL-4 expression was detected by qRT-PCR analysis. The data was expressed as the mean of three independent experiments. Wilcoxon test was used to calculate the P -value. P < 0.05. ( D ) CD4 naive cells were cultured in the conditioned medium for Th2 development and transfected with s30840 or control. After 7 days, the Th2 T-lymphocytes (intracellular stained as IL-4 + IFN-gamma - ) were detected with FACS analysis. The data was shown as a mean of six independent experiments. P < 0.01. ( E ) The CD4 naive cells were cultured in a conditioned medium for Th2 development with or without the transfection of inhibitor. We adoptively transferred the naive cells into the irradiated NOG mice which were first humanized. After 7 days, Th2 T-lymphocytes (intracellularly stained as IL-4 + IFNgamma - ) were detected by using FACS analysis. The data were expressed as the mean of six independent experiments. Statistical significance between two samples was determined by using the student's t -test. P < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. piR30840 significantly decreases IL-4 expression. ( A and B ) s30840 decreases IL-4 expression in purified CD4 T lymphocytes from PBMCs. IL-4 expression was detected by qRT-PCR (left panel), northern blotting (right panel) (A) and western blotting (B). The data represents three independent experiments. Con represents the synthesized random sequence for control. s30840 represents the synthesized piR30840. P < 0.05. ( C ) s30840 decreases IL-4 expression in activated CD4 T lymphocytes. IL-4 expression was detected by qRT-PCR (upper panel) and western blotting (lower panel) analyses. The data represents three independent trials. P < 0.05. ( D ) Antisense inhibitor piR30840 antisense upregulates IL-4 in purified CD4 T lymphocytes from PBMCs. IL-4 expression was detected by qRT-PCR (left panel) and western blot (right panel) analyses. The data represents three independent trials. P < 0.05. ( E and F ) The construct containing all the IL-4 exons and introns (construct pIL4) was co-transfected with s30840 into 293T cells. IL-4 expression was detected by qRT-PCR (left panel) and western blotting (right panel) (E), as well as by northern blotting (F) analyses. The data represents three independent experiments. P < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. piR30840 downregulates IL-4 pre-mRNA via binding to its intron region. ( A ) The putative binding sites of piR30840 in the introns of IL-4 pre-mRNA. ( B - D ) IL-4 constructs containing the first (In1) (B), second (In2) (C) or third intron (In3) (D) were co-transfected with s30840 into 293T cells respectively. IL-4 expression was then analyzed by qRT-PCR (upper panel) and western blotting (lower panel). Statistical significance between two samples was determined by using t -test. P < 0.01. ( E ) The construct In2 was co-transfected with s30840 or control. The RNA expression of IL-4 was analyzed by northern blotting. ( F ) The construct In2m was co-transfected with s30840. IL-4 expression was examined by qRT-PCR and western blotting analyses. ( G ) The construct In2m was co-transfected with the piR30840 mutation (30840mut). IL-4 expression was determined by qRT-PCR and western blotting analyses.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. TRAMP complex and exosome are involved in the decay of IL-4 pre-mRNA in the nucleus. ( A ) s30840 was co-transfected with siRNAs specific for trf4, air or mtr4 into CD4 T lymphocytes, respectively. The IL-4 expression was analyzed by western blotting. The data represents three independent experiments. ( B ) The interaction between Piwil4 and Mtr4 in 293T cells was detected by co-immunoprecipitation. Actin was used as loading control. ( C ) Piwil4 interacting with Prp4 was detected by co-immunoprecipitation in 293T cells. ( D ) s30840 was co-transfected with siRNA specific for rrp6, rrp44 and rrp42 into primary CD4 T-lymphocytes. The expression of IL-4 was detected by western blotting. The data represents three independent trials.

Supportive validation

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Experimental details: Figure 5 Blockade of transforming growth factor (TGF)-beta I-III did in part prevent regulatory T cells (Treg) induction. ( A ) CFSE-labeled CD4 + CD25 - T cells were cocultured with platelets in the ratio of 1:50 and were stimulated with anti-CD3 mAb (0.5 ug/mL) and anti-CD28 mAb (1.0 ug/mL) in the presence of either anti-TGF-beta I-III (10 ug/mL) or anti-TGF-beta receptor II (10 ug/mL) antibodies. Antibodies were added at day 0. The expression of Foxp3 and GARP and cell proliferation were determined on day 3 via flow cytometry. ( B ) Production of IL-2 and IFN-gamma was assessed by intracellular flow cytometry on day 6. The graphs show cells cultured in the presence of platelets normalized to CD4 + CD25 - T cells without platelets. Dot plots show one representative result of 10 independent experiments ( n = 10, box and whiskers, medians +- min/max, * p < 0.05, ** p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Combining blockade of TGF-beta signaling and GARP led to a complete inhibition of platelet effects. ( A ) CFSE-labeled CD4 + CD25 - T cells were cocultured with platelets in the ratio of 1:50 and were stimulated with anti-CD3 mAb (0.5 ug/mL) and anti-CD28 mAb (1.0 ug/mL). CD4 + CD25 - T cells were incubated for 15 min with TGF-beta receptor II (10 ug/mL) antibody prior to coculture, as indicated. Excess antibody was removed. Pre-treated CD4 + CD25 - T cells were cultured in the presence of either anti-TGF-beta I-III (10 ug/mL) and/or anti-GARP Ab (10 ug/mL) antibodies. Antibodies were added at day 0. The expression of Foxp3, GARP and cell proliferation were determined on day 3 via flow cytometry. ( B ) Production of IL-2 and IFN-gamma was assessed by intracellular flow cytometry on day 6. The graphs show cells cultured in the presence of platelets normalized to CD4 + CD25 - T cells without platelets. Dot plots show 1 representative result of 10 independent experiments ( n = 3, means +- SD, * p < 0.05, ** p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Platelet-conditioned medium (PCM) inhibited IFN-gamma production, but failed to induce a Treg phenotype. ( A ) CD4 + CD25 - T cells were cultured in X-Vivo 15 (Lonza, Basel, Switzerland) with 60% PCM content, with or without 10 ug/mL anti-GARP Ab and stimulated with 0.5 ug/mL anti-CD3 mAb and 1.0 ug/mL anti-CD28 mAb. Antibodies were added at day 0. The expression of Foxp3, GARP and cell proliferation were determined at day 3 with flow cytometry. ( B ) Cytokine production of IL-2 and IFN-gamma was measured by intracellular flow cytometry on day 6. Dot plots show one representative result of five independent experiments ( n = 5, box and whiskers, medians +- min/max, * p < 0.05, ** p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure A4 Thrombin-activated platelets induced a regulatory phenotype in CD4 + CD25 - T cells. 1 x 10 6 CD4 + CD25 - T cells were stimulated with 0.5 ug/mL anti-CD3 mAb and 1.0 ug/mL anti-CD28 mAb with or without 50 x 10 6 platelets for 6 days and treated with or without 10 U/mL thrombin. ( A ) Foxp3 and GARP expression and proliferation were determined at day 3 via flow cytometry. ( B ) Using intracellular flow cytometry, we analyzed cytokine production of IL-2 and IFN-gamma on day 6. Dot plots show one representative result of five independent experiments ( n = 5, box and whiskers, medians +- min/max, * p < 0.05, ** p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure A5 TRAP-6-activated platelets induced a regulatory phenotype in CD4 + CD25 - T cells. 1 x 10 6 CD4 + CD25 - T cells were stimulated with 0.5 ug/mL anti-CD3 mAb and 1.0 ug/mL anti-CD28 mAb with or without 50 x 10 6 platelets for 6 days and treated with or without 5 uM TRAP-6. ( A ) Foxp3 and GARP expression and proliferation were determined at day 3 via flow cytometry. ( B ) Using intracellular flow cytometry, we analyzed cytokine production of IL-2 and IFN-gamma on day 6. Dot plots show one representative result of five independent experiments ( n = 5, box and whiskers, medians +- min/max, * p < 0.05, ** p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Comparison of proliferation and cytokine production in naive cord blood T cells (CBTC) in Protein Kinase C zeta (PKCzeta) low or high group. ( a ) Comparison of PKCzeta between cord blood (CB) and adult blood (AB) T cells, n = 24 for each AB or CB. ( b ) Shows lymphoproliferation as a Stimulation index (SI) and disintegrations per minute (DPM) in 3 H-thymidine pulsed cultures stimulated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA) ( c ) Purified CBCTs were stained with Carboxyfluorescein succinimidyl ester (CFSE) dye and stimulated with immobilized anti-CD3/-CD28 antibodies for 3 days. Gating and representative histogram for CFSE dilution after exclusion of doublets and dead cells. Overlaid histograms for stained unstimulated and unstained stimulated samples were used as control and for gating the non-proliferating cells and for auto-fluorescence, respectively. ( d ) Naive CB CD3 + T cells were stimulated with PHA/PMA (18 h) and percentage of CD3 + T cells producing interleukin-4 (IL-4) and Interferon-gamma (IFN-gamma) and median fluorescent intensity (MFI) were examined by flow cytometry assays. ( e ) On day 5 of CFSE stained culture (anti-CD3/-CD28), cells were re-stimulated with PHA/PMA (18 h) for detection of intracellular cytokine. Representative flow dot plots and data for CFSE dye dilution and IFN-gamma producing cells in high and low PKCzeta group. Data mean +- SD of n = 3 for of low and n = 4 high PKCzeta group. ** p < 0.01. ns: not sig

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3 Changes in the percentages and absolute numbers of Th1 and Th2 cells in SFTS patients. (A): Th1 cells (CD4 + IFN-gamma+) and Th2 cells (CD4 + IL-4+) by flow cytometry in the healthy controls, surviving SFTS patients, and deceased SFTS patients, as defined by flow cytometry. The cells were gated on the CD3 + CD4+ population within the single-cell lymphocyte gate. (B), (D): The percentages and numbers of Th1 and Th2 cells in the healthy controls (n = 11) and the surviving patients with SFTS in the acute phase (n = 30) and SFTS in the recovery phase (n = 30). (C), (E): The percentages and numbers of Th1 and Th2 cells at admission in the surviving patients (n = 30) and the deceased patients (n = 12). (F), (G): Dynamic changes in the percentages and numbers of Th1 and Th2 cells in the surviving patients (n = 30) and the deceased patients (n = 12). These parameters were monitored at indicated time points for the entire hospital stay of the patients, and the dashed line represents the median of the uninfected controls. The data are shown as the median +- 95% CI. Statistical analysis was performed using the Mann-Whitney U test or the Wilcoxon matched pair test. The level of significance is indicated as follows: ns, not significant; *p < .05; **p < .01; ***p < .001; ****p < .0001; Fig. 3