Fig 1: TAGAP is important for T helper cell polarization in mice.a Cells of lymph nodes and spleens from heterozygous control or TAGAP-deficient mice were analyzed by flow cytometry by gating on CD4+, and analyzed by IL-17A or IFN-γ-producing cells. Right panel indicates quantitative result, and the top right panel represents data from spleen, and the bottom right data represent data from lymph nodes (n = 8). b Heterozygous control or TAGAP-deficient mice were immunized subcutaneously with 200 μg MOG35–55 and 400 μg Mycobacteria tuberculosis H37RA in 200 μL of complete Freund’s adjuvant (Difco). Lymph nodes were collected 10 days later and single-cell suspensions were prepared. Cells were cultured with MOG35–55 (20 μg/mL) for another 3 days, followed by flow cytometry analysis of indicated cell populations. Right panel were quantitative results (n = 4). c Heterozygous control or TAGAP-deficient mice were immunized as in b, except for immunization with 200 μg MOG35–55 plus CFA and 400 μg heat-killed C. albicans (sc-5314). Right panel were quantitative results (n = 4). d Heterozygous control or TAGAP-deficient mice were immunized as in b, except for immunization with 200 μg MOG35–55 plus CFA only (n = 4). ns: P = 0.5226 and P = 0.288. e Naive CD4+ T cells were isolated from spleens of heterozygous control or TAGAP-deficient mice, and polarized in vitro for 3 days in the Th17 or Th1 cell polarization conditions. Cells were analyzed by flow cytometry by gating on CD4+, and analyzed by IL-17A or IFN-γ-producing cells. Right panel were quantitative results (n = 10). ns: P = 0.1848 and P = 0.9952. f Wild-type mice were injected intravenously with 2 × 105 live C. albicans (sc-5314) in 100 μL PBS. Ten days later, CD4+ T cells were isolated from spleen, and co-cultured with DCs isolated from heterozygous control or TAGAP-deficient mice for another 3 days together with indicated numbers of heat-killed C. albicans. Cells were analyzed by flow cytometry by gating on CD4+, and analyzed by IL-17A or IFN-γ-producing cells (n = 6). Right panel were qualified results. *P < 0.05; **P < 0.01; ***P < 0.001 based on two-sided unpaired t test a–f. All error bars represent SEM of technical replicates. Data are representative of two independent experiments.
Fig 2: TAGAP expression level correlates positively with Th17 cell abundance in humans.a Human PBMCs were divided in different groups based on the TAGAP SNP genotypes, and TAGAP mRNA expression was analyzed by RT and real-time PCR. Each dot in the data represent the data from each individual (n = 17, 10, 11 for rs1738074; n = 12, 13, 15 rs3127214). b, c Human PBMCs of TAGAP rs1738074 T/T and C/C b or TAGAP rs3127214 C/C and T/T c were analyzed by flow cytometry by gating on CD3+, and analyzed by IL-17A or IFN-γ-producing cells. Lower panel were quantitative results. d, e PBMCs of TAGAP different genotypes were stimulated with Curdlan (100 μg/ml) or α-Mannan (100 μg/ml) for 3 h, followed by real-time PCR analysis of indicated genes. *P < 0.05; **P < 0.01; ***P < 0.001 based on two-sided unpaired t test a–e. All error bars represent SEM of technical replicates.
Fig 3: TAGAP-binding partner EPHB2 is important for antifungal signaling activation.a HEK293T cells were transfected with indicated plasmids, and cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis for indicated proteins. b Flag-TAGAP stable transfected THP-1 cells were differentiated with PMA (25 ng/mL) for 3 days, followed by immunoprecipitation by Flag antibody. Protein elution was analyzed by mass spectrometry analysis. Graph represents mass spectrometry result of TAGAP-interacting proteins. c Human THP-1 cells were non-polarized or polarized by adding PMA (25 ng/mL) for 3 days, and were stimulated with heat-killed C. albicans (MOI = 2) for 0, 15, and 30 min, followed by western blot analysis of indicated proteins. d, e HEK293T cells were transfected with indicated plasmids, and cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis for indicated proteins. f Control siRNA or EPHB2 siRNA-transfected BMDMs were stimulated with Curdlan (100 μg/ml) for 3 h, followed by real-time PCR analysis of indicated genes. g–i THP-1 cells infected with control gRNA or EPHB2-gRNA were stimulated with heat-killed C. albicans (MOI = 2), Curdlan (100 μg/ml) or α-Mannan (100 μg/ml) for the indicated times, followed by western blot analysis of indicated proteins. j THP-1 cells infected with control gRNA or EPHB2-gRNA were stimulated with heat-killed C. albicans (MOI = 2) for the indicated times, followed by real-time PCR analysis for indicated genes. P < 0.05; **P < 0.01; ***P < 0.001 based on two-sided unpaired t test f, j. Real-time PCR data of f and j were collected from two independent experiments. All error bars represent SEM of technical replicates. Data are representative of three independent experiments.
Fig 4: TAGAP is required for Dectin-2/3 and mincle ligands-induced signaling activation.a BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with α-Mannan (100 μg/ml) for 3 and 6 h, followed by real-time PCR analysis of indicated genes. b BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with α-Mannan (100 μg/ml) for indicated times, followed by western blot analysis of indicated protein expression. c Control- gRNA or TAGAP-gRNA knocked down THP-1 cells were stimulated with α-mannan (100 μg/ml) for indicated times, followed by western blot analysis of indicated proteins. d BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with TDB (50 μg/well) for 3 and 6 h, followed by real-time PCR analysis of indicated genes. e BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with TDB (50 μg/well) for indicated times, followed by western blot analysis of indicated protein expression. *P < 0.05; **P < 0.01; ***P < 0.001 based on two-sided unpaired t test a, d. All error bars represent SEM of technical replicates. Data are representative of three independent experiments.
Fig 5: TAGAP is required for Dectin-1 ligand-induced signaling activation.a Real-time PCR was done from different organs (upper panel) and cell types (lower panel) from three mice, and the result was shown. b, c BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with Curdlan (100 μg/ml) for the indicated times, followed by western blot or real-time PCR analysis of indicated proteins and gene expression. d BMDMs from heterozygous control or TAGAP-deficient mice were stimulated with heat-killed C. albicans sc-5314 (upper panel, MOI = 2) or d-zymosan (lower panel, 100 μg/ml) for the indicated times, followed by western blot analysis of indicated proteins. e BMDMs from heterozygous control or TAGAP-deficient mice were stimulated with heat-killed C. albicans sc-5314 for 3 and 6 h, followed by real-time PCR analysis of indicated gene expression. f Control- gRNA or TAGAP-gRNA knocked down THP-1 cells were stimulated with heat-killed C. albicans sc-5314 (MOI = 2) for indicated times, followed by western blot analysis of indicated proteins. g BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with heat-killed C. albicans sc-5314 for indicated times, followed by western blot analysis of indicated proteins. h BMDMs from heterozygous control mice or TAGAP-deficient mice were stimulated with Curdlan (100 μg/ml), followed by RNA-Seq analysis of gene expression. Heat-map of RNA-Seq data was shown. Arrow indicated top-changed gene expression in cytokine and chemokine groups. The scale bar representing fold induction was shown. *P < 0.05; **P < 0.01; ***P < 0.001 based on two-sided unpaired t test for panel a, c, e. All error bars represent SEM of technical replicates. Data are representative of three independent experiments except for h.
Supplier Page from Abcam for Anti-TAGAP antibody [EPR15593]