stat6 (rabbit ab) antibody  (Cell Signaling Technology Inc)

Journal: PLOS Pathogens

Article Title: Th2-dependent STAT6-regulated genes in intestinal epithelial cells mediate larval trapping during secondary Heligmosomoides polygyrus bakeri infection

doi: 10.1371/journal.ppat.1011296

Figure Lengend Snippet: Bone marrow derived macrophages (BMDM, purity >95%) were generated and splenic B cells (purity > 90%) isolated from mice that do express STAT6 only under control of the LysMCre promoter (Mac-STAT6), STAT6 knockout mice (STAT6ko) and BALB/c wild-type (WT) controls. A) Western blot of unstimulated (w/o) and IL-4-stimulated BMDM and B cell lysates of the indicated genotypes for phosphorylated STAT6 (pSTAT6), total STAT6 and β-actin as loading control. Western blot is representative for three to five mice per genotype from two independent experiments. B) qRT-PCR of BMDM from either Mac-STAT6, STAT6ko or WT mice stimulated with 20 ng/ml IL-4 for 48 hrs. Bars show mean expression + SEM of Arg1 , Retnla and Pdcd1lg2 normalized on Hprt of three to four mice per genotype out of two experiments. Statistical significance was determined by Two-Way-ANOVA with Holm-Sidak post-hoc testingon log-transformed data. ***p < 0.001; *p < 0.05.

Article Snippet: Membranes were blocked with 3% BSA in TRIS-buffered saline-tween buffer (TBS-T) and phospho-STAT6 (Y641), STAT6 (Rabbit Ab) or beta-actin (13E5) were applied as primary antibodies (all Cell Signaling Technology, Danvers, MA) at 4°C overnight.

Techniques: Derivative Assay, Generated, Isolation, Control, Knock-Out, Western Blot, Quantitative RT-PCR, Expressing, Transformation Assay

Journal: PLOS Pathogens

Article Title: Th2-dependent STAT6-regulated genes in intestinal epithelial cells mediate larval trapping during secondary Heligmosomoides polygyrus bakeri infection

doi: 10.1371/journal.ppat.1011296

Figure Lengend Snippet: A) Schematic timeline: Mac-STAT6 mice, STAT6ko mice or BALB/c (WT) control mice were primary infected with Hpb , infection was cleared by Pyrantel pamoate after two weeks and four to six weeks later, mice were secondarily infected. 5 μg/mouse IL-4c in 100 μl PBS or PBS for control groups was injected i . p . on days 1, 3, 5 and 7 after secondary infection. B) Mean + SEM of granuloma with or without (w/o) larvae on day 9 after secondary infection. Statistical analysis was performed separately for granuloma with or w/o larvae by Kruskal-Wallis with Dunn’s post-hoc testing. *p<0.05. Data are pooled from three independent experiments with five to seven mice per group. C and D) Histological stainings and quantifications of small intestinal cross-sections day 9 after secondary Hpb infection for detection of Relm-α (red) and CD68 (green) (C), or DAPI (blue) and Arg1 (green) (D). Quantification plots display Mean + SEM of percentage of Relm-α + or Arg1 + of DAPI + CD68 + macrophages, respectively. Representative picture and quantification for four to seven mice per genotype. Scale bar is 100 μm.

Article Snippet: Membranes were blocked with 3% BSA in TRIS-buffered saline-tween buffer (TBS-T) and phospho-STAT6 (Y641), STAT6 (Rabbit Ab) or beta-actin (13E5) were applied as primary antibodies (all Cell Signaling Technology, Danvers, MA) at 4°C overnight.

Techniques: Control, Infection, Injection

Journal: PLOS Pathogens

Article Title: Th2-dependent STAT6-regulated genes in intestinal epithelial cells mediate larval trapping during secondary Heligmosomoides polygyrus bakeri infection

doi: 10.1371/journal.ppat.1011296

Figure Lengend Snippet: A) Schematic timeline for the CD4 depletion experiment performed with mice with constitutively active STAT6 in IECs (VillinCre_STAT6vt) and Cre - littermate controls (STAT6vt): Primary Hpb infection was cleared by oral gavage with twice 1 mg Pyrantel pamoate per mouse. 4–6 weeks later, mice were secondarily infected with Hpb and anti-CD4 or isotype antibody (AB) was injected i . v . on day 0 and 4 of secondary infection. B) Number of counted granuloma with or without (w/o) larvae in the submucosa on day 9 or 10 after secondary Hpb infection. Bars show mean count + SEM of four to eleven mice per group from two independent experiments. C) Eggs per gram feces counted on day 14 after secondary Hpb infection. Data show mean count + SEM of three to seven mice per group from two independent experiments. D) Relative expression of Arg1 , Retnla , Retnlb , Chil3 , Pdcd1lg2 , and Mrc1 , calculated by normalisation to Hprt . Bars show mean + SEM of four to eleven mice per group from two independent experiments. E) Histological staining and quantification of Relm-α (red) and CD68 (green) in tissue sections from small intestine day 9 after secondary Hpb infection in the left panel and for DAPI (blue) and Arg1 (green) in the right panel. Mean + SEM of percentage of Relm-α + or Arg1 + of DAPI + CD68 + cells, respectively. Representative picture for one mouse per genotype and quantification for all five to ten mice per group from two independent experiments. Scale bar corresponds to 100 μm. B-E) Statistical significance was determined by Two-Way ANOVA with Holm-Sidak post-hoc testing. ***p < 0.001; **p < 0.01; *p < 0.05.

Article Snippet: Membranes were blocked with 3% BSA in TRIS-buffered saline-tween buffer (TBS-T) and phospho-STAT6 (Y641), STAT6 (Rabbit Ab) or beta-actin (13E5) were applied as primary antibodies (all Cell Signaling Technology, Danvers, MA) at 4°C overnight.

Techniques: Infection, Injection, Expressing, Staining

Journal: The Journal of Clinical Investigation

Article Title: MicroRNA-210 overexpression promotes psoriasis-like inflammation by inducing Th1 and Th17 cell differentiation

doi: 10.1172/JCI97426

Figure Lengend Snippet: (A) miR-210 target sequences (in red) of the 3′-UTR of STAT6 (A, upper panel) and LYN (A, lower panel) mRNAs and corresponding mutant sequences, which were included in luciferase reporter vectors. (B) Luciferase activities were measured in HEK293T cells cotransfected with luciferase reporter vectors and agomir-210 or agomir-NC, n = 3 per group. (C) Normal CD4+ T cells were transfected with agomir-210 or agomir-NC, and psoriatic CD4+ T cells were transfected with antagomir-210 or antagomir-NC. STAT6 (upper panel) and LYN (lower panel) protein levels were analyzed. (D) The protein levels of STAT6 and LYN in splenic CD4+ T cells from IMQ-treated WT and KO mice. (E–H) STAT6 and LYN protein levels in CD4+ T cells (E and F) and skin lesions (G and H) derived from psoriasis patients and IMQ-induced psoriasis-like mouse models (BALB/c) as well as their healthy controls. Data (B and C) are representative of at least 3 independent experiments. Data represent the mean ± SEM. *P < 0.05, **P < 0.01. NS, not significant. Two-tailed unpaired Student’s t test (B) was used.

Article Snippet: Rabbit anti–HIF-1α Ab (1:1,000; Cell Signal Technology, catalog 14179), rabbit anti-STAT6 Ab (1:1,000; Cell Signal Technology, catalog 9362), mouse anti-LYN Ab (1:1,000; Abcam, catalog ab1890), mouse anti–β-actin Ab (1:2,000, Abcam, catalog ab6276) or goat anti-GAPDH Ab (1:2,000, Abcam, catalog ab9483) was used.

Techniques: Mutagenesis, Luciferase, Transfection, Derivative Assay, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: MicroRNA-210 overexpression promotes psoriasis-like inflammation by inducing Th1 and Th17 cell differentiation

doi: 10.1172/JCI97426

Figure Lengend Snippet: (A and B) Normal human CD4+ T cells were transfected with STAT6 siRNAs and overexpression plasmid (A, n = 3) or LYN siRNAs and overexpression plasmid (B, n = 3) separately for 48 hours. STAT6 and LYN protein levels (left panel), and the mRNA levels of IL17A, IL17F, IFNG, and IL4 (middle and right panels) were detected in transfected cells. (C and D) Normal human CD4+ T cells were transfected with agomir-210 or agomir-NC, and after 24 hours, the STAT6 overexpression plasmid or plasmid control (C, n = 3) and LYN overexpression plasmid or plasmid control (D, n = 3) were transfected into cells. Then, cells were collected to detect the protein levels of STAT6 or LYN (left panel) and the mRNA levels of IL17A, IL17F, IFNG, and IL4 (right panel) at 48 hours after transfection. Similar results were obtained from 3 independent experiments. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. Two-tailed unpaired Student’s t test (A and B) or 1-way ANOVA with Bonferroni’s post hoc test (C and D) was used.

Article Snippet: Rabbit anti–HIF-1α Ab (1:1,000; Cell Signal Technology, catalog 14179), rabbit anti-STAT6 Ab (1:1,000; Cell Signal Technology, catalog 9362), mouse anti-LYN Ab (1:1,000; Abcam, catalog ab1890), mouse anti–β-actin Ab (1:2,000, Abcam, catalog ab6276) or goat anti-GAPDH Ab (1:2,000, Abcam, catalog ab9483) was used.

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: MicroRNA-210 overexpression promotes psoriasis-like inflammation by inducing Th1 and Th17 cell differentiation

doi: 10.1172/JCI97426

Figure Lengend Snippet: (A and B) The expression of miR-210 in normal human CD4+ T cells stimulated with IL-23, TGF-β, IL-6, or IL-1β (A, n = 3 per group). The protein levels of HIF-1α, STAT6, and LYN in cells stimulated with IL-23 or TGF-β (B). (C) The miR-210 expression levels in human CD4+ T cells transfected with HIF-1α siRNA or siRNA controls followed by stimulation with IL-23 or TGF-β for 24 hours (n = 3 per group). (D) CD4+ T cells were transfected with HIF-1α overexpression plasmid (upper panel) or were stimulated with TGF-β (lower 2 panels). Co-IP and Western blot showed the interaction between HIF-1α and P300. (E–G) The enrichment of HIF-1α, P300, and H3ac on the miR-210 promoter in psoriatic CD4+ T cells transfected with HIF-1α siRNA or controls (E, n = 3), normal CD4+ T cells transfected with HIF-1α plasmid or controls (F, n = 3), or normal CD4+ T cells stimulated with TGF-β (G, n = 3). Data (B and D) are representative of at least 3 independent experiments. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with Dunnett’s post hoc test (A and G) or 2-tailed unpaired Student’s t test (C, E, and F) was used.

Article Snippet: Rabbit anti–HIF-1α Ab (1:1,000; Cell Signal Technology, catalog 14179), rabbit anti-STAT6 Ab (1:1,000; Cell Signal Technology, catalog 9362), mouse anti-LYN Ab (1:1,000; Abcam, catalog ab1890), mouse anti–β-actin Ab (1:2,000, Abcam, catalog ab6276) or goat anti-GAPDH Ab (1:2,000, Abcam, catalog ab9483) was used.

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: MicroRNA-210 overexpression promotes psoriasis-like inflammation by inducing Th1 and Th17 cell differentiation

doi: 10.1172/JCI97426

Figure Lengend Snippet: In psoriasis patients, inflammatory factors TGF-β and IL-23 stimulate the expression of HIF-1α, which recruits P300 to the miR-210 promoter region and increases the H3ac levels on miR-210 promoters, thus leading to the upregulation of miR-210 expression. miR-210 represses STAT6 and LYN expression through binding the 3′-UTRs of STAT6 and LYN mRNA, which promotes Th1/Th17 cell differentiation and inhibits Th2 cell differentiation in psoriasis. Moreover, miR-210 overexpression also enhances the proliferation and chemokine secretion of keratinocytes, which increases the recruitment of activated T cells in skin lesions. Together, the effects of the increased miR-210 in CD4+ T cells and keratinocytes contribute to the formation of psoriatic skin lesions.

Article Snippet: Rabbit anti–HIF-1α Ab (1:1,000; Cell Signal Technology, catalog 14179), rabbit anti-STAT6 Ab (1:1,000; Cell Signal Technology, catalog 9362), mouse anti-LYN Ab (1:1,000; Abcam, catalog ab1890), mouse anti–β-actin Ab (1:2,000, Abcam, catalog ab6276) or goat anti-GAPDH Ab (1:2,000, Abcam, catalog ab9483) was used.

Techniques: Expressing, Binding Assay, Cell Differentiation, Over Expression