GIFT4, GM-CSF, IL-4 and IFN-γ were quantified with ELISA kits (eBiosciences, San Diego, CA). GIFT4 were profiled by Western blot (WB) with anti-GM-CSF or anti-IL-4 antibodies (R&D systems). Murine B-cells (106 cells/ml) treated with GIFT4, GM-CSF and IL-4 with or without JAK inhibitors were lysed with buffer as described (21 (link)). STAT phosphorylation in B-cells was determined by WB with anti-pSTAT1 (Tyr701, D4A7), anti-pSTAT3 (Tyr705, D3A7), anti-pSTAT5 (Tyr694, D47E7), anti-pSTAT6 (Tyr641, C11A12), or anti-STAT antibodies (Cell Signaling). For immunoprecipitation of GMCSFRβ by common γc receptor after GIFT4 or GM-CSF and IL-4 stimulation, the B-cells were lysed with buffer as described (21 (link)). One mg of protein per sample was incubated with 2 mg of anti-common γc or anti-GMCSFR antibodies, or IgG isotype control (Santa Cruz Biotechnology, CA) overnight at 4°C. The sample-antibody complexes were incubated with protein A agarose for 2 hour at 4°C. The bound proteins were eluted for WB with anti-GMCSFR or anti-IL4R antibodies.
Anti p stat6
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p-STAT6 is a primary antibody product that can be used to detect the phosphorylated form of STAT6 transcription factor. STAT6 is a key signaling molecule involved in the IL-4 and IL-13 signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti stat6, by Cell Signaling Technology (9 mentions)
Anti p stat1, by Cell Signaling Technology (3 mentions)
Anti p stat3, by Cell Signaling Technology (2 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
Anti stat3, by Cell Signaling Technology (2 mentions)
Anti stat1, by Cell Signaling Technology (2 mentions)
Anti pparγ, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti p jak1, by Cell Signaling Technology (2 mentions)
Anti jak1, by Cell Signaling Technology (2 mentions)
Anti p jak2, by Cell Signaling Technology (2 mentions)
Anti jak2, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti p stat5, by Cell Signaling Technology (1 mentions)
Anti gm csf, by R&D Systems (1 mentions)
Isotype control igg, by Santa Cruz Biotechnology (1 mentions)
Anti il 4 antibody, by R&D Systems (1 mentions)
15 protocols using anti p stat6
1
GIFT4 Signaling and B-cell Activation
2
Multiparametric Flow Cytometry for Immune Signaling
3
Detailed Protocol for Inflammatory Signaling
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Acetonitrile (34998) and formic acid (00940) for LC-MS were obtained from Merck (WGK, Germany). InComplete Freund’s adjuvant (IFA), Complete Freund’s adjuvant (CFA), and bovine type II collagen were obtained from Chondrex, Inc. (Redmond, Wash.). ELISA kits for detections of IL-1β (10 × 96t, 88-7013-88), IL-6 (2 × 96t, 88-7064-86), TNF-α (2 × 96t, 88-7324-22), IL-4 (2 × 96t, 88-7044-22) and IL-10 (2 × 96t, 88-7105-22) were purchased from Invitrogen (Karlsruhe, Germany). Anti-IKKα (ab32041, 1:1000), Anti-IKKβ (ab124957, 1/1000), anti-IKKα/β (ab194528, 1:1000), anti-NF-κB p65 antibody (ab16502, 1:1000), anti-P-NF-κB p65 (phospho S536) antibody (ab76302, 1:1000), anti-P38 (ab122517, 1:1000) antibody, anti-P-P38 (phospho Y182) antibody (ab47363, 1:1000), anti-Histone H3 antibody (ab1791, 1:1000) and secondary antibodies were obtained from Abcam (Cambridge, United Kingdom). Anti-IκBα, anti-P-IκBα (phospho S36/32), anti-P-ERK (Thr202/Tyr204), Anti-ERK, anti-JNK, anti- P- JNK (Thr183/Tyr185), anti-STAT6 and anti-P-STAT6 antibodies were obtained from cell signaling technology (Boston, United States). All other reagents and chemicals used were of standard biochemical quality.
4
Kupffer Cell Co-Immunoprecipitation Assay
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Primary Kupffer cells from male C57BL/6J mice (8–10 weeks) were isolated according to the method described. The co‐IP experiment was performed using the Thermo Scientific Pierce co‐IP kit (Thermo Scientific, Rockford, USA) according to the manufacturer's introduction. The co‐IP samples were analyzed by western blotting using anti‐PXR (Santa Cruz Biotechnology, CA, USA) and anti‐p‐STAT6 (Cell Signaling Technology, Danvers, USA). Anti‐rabbit or anti‐mouse IgG was purchased from Abclonal, Wuhan, China).
5
Western Blot Analysis of Immune Markers
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Protein extracts were subjected to SDS-PAGE (8–12% gels) and blotted onto PVDF membranes. After blocking with 5% fat-free milk, the membranes were incubated with the following antibodies: anti-AKT, anti-p-AKT, anti-Arg-1, anti-GAPDH, anti-β-actin (all purchased from Santa Cruz Biotechnology), anti-STAT6, anti-p-STAT6, anti-STAT3, anti-p-STAT3(705) (all purchased from Cell Signaling Technology), anti-Ym1, anti-IL10 (all purchased from Abcam) at 4 °C overnight. The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated IgG (MULTI Sciences) and visualized with enhanced chemiluminescence (ECL, PerkinElmer) detection reagents (Thermo scientific, USA). β-actin or GAPDH was used as a loading control. IL-10 concentration in plasma, tissues, and cell culture supernatants were determined by ELISA kit (DAKEWE). The tissue ELISA measurements were normalized to the protein content of the homogenates.
6
Western Blot Analysis of Intestinal Tight Junctions
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BMDMs or ileal tissues were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). A bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) was used to determine the protein concentration. Approximate 30 μg of protein was separated by 10% SDS-PAGE and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 1% BSA/Tris-buffered saline (TBS), the blots were then incubated with primary antibodies at 4°C overnight. The following primary antibodies obtained from Cell Signaling Technology were used in this study: anti-β-actin (Cat. no. 3700; 1:1,000), anti-PPARγ (Cat. no. 2435; 1:1,000), anti-claudin-1 (Cat. no. 13255; 1:2,000), anti-occludin (Cat. no. 91131; 1:1,500), anti-ZO-1 (Cat. no. 13663; 1:1,000), anti-STAT1 (Cat. no. 14994; 1: 3,000), anti-p-STAT1 (Tyr701, Cat. no. 9167; 1:1,000), anti-STAT6 (Cat. no. 5397; 1: 2,000), and anti-p-STAT6 (Tyr641, Cat. no. 9361; 1:1,500). The membranes were incubated with corresponding HRP-conjugated secondary antibody at room temperature for 30 min. The signals were visualized by Pico Plus enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific) and analyzed by Quantity One software (Bio-Rad, CA, USA). β-Actin served as the loading control.
7
Immunoblotting Analysis of Inflammatory Markers
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All cell lysates were separated by 10% or 12% SDS-polyacrylamide gel electrophoresis. Then, they were transferred to PVDF membranes. Blocking with 5% skimmed milk powder (BD Pharmingen) for 1 h at room temperature, the membranes were incubated with specific anti-Arg1 (1: 1,000), anti-TNFα (1: 500), anti-IL1β (1: 1,000), anti-TNFSF15 (1: 1,000) from Abcam, anti-p-p38 MAPK (1: 1,000), anti-p38 MAPK (1: 1,000), anti-p-JNK (1: 1,000), anti-JNK (1: 1,000), anti-p-Erk1/2 (1: 2,000), anti-Erk1/2 (1: 2,000), anti-p-Akt (1: 1,000), anti-Akt (1: 1,000), anti-p-STAT1 (1: 1,000), anti-STAT1 (1: 1,000), anti-p-STAT6 (1: 1,000), anti-STAT6 (1: 1,000), anti-p-STAT3 (1: 1,000), anti-STAT3 (1: 1,000), anti-GADPH (1: 2,000) from Cell Signaling Technology, anti-iNOS (1: 1,000, Introvigen) or anti-β-actin (1: 2,000, ZSGB-BIO) overnight at 4°C. Then the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Then, protein bands were visualized by ECL Western blot reagent (Bioworld Technology).
8
Investigating Cell Migration Mechanisms
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Anti-RSU-1 rabbit polyclonal antibody for immunoblotting was kindly provided by Dr. Mary Lou Cutler, Professor at the Uniformed Services University of the Health Sciences, Bethesda USA. Anti-pSTAT6 and anti-STAT6 were obtained from Cell Signaling. Anti-MMP13 was purchased from Abcam. Phospho-STAT6 inhibitor, AS1517499, was obtained from Axon Medchem. RSU-1 siRNA was purchased from Santa Cruz Biotechnology. Rhodamine-Phalloidin was obtained from Biotium and 4′,6-Diamidino-2-Phenylindole (DAPI) was obtained from Roche. Transwell inserts were purchased from Greiner Bio-One and Matrigel as well as Collagen I was obtained from Corning. QIAzol Lysis Reagent was purchased from QIAGEN.
9
Quantifying JAK-STAT Signaling in Mouse Lungs
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The total proteins from mice lung tissues were isolated, and the protein concentrations were determined using a bicinchoninic acid assay. The proteins from whole-cell lysates were used for western blot. Anti-JAK1, anti-p-JAK1, anti-JAK2, anti-p-JAK2, anti-STAT6, anti-p-STAT6, and GAPDH (all purchased from Cell Signaling Technology, Leiden, the Netherlands) antibodies were used. Densitometry of bands from western blots was measured using ImageJ software (National Institutes of Health, Bethesda, USA), and the density of the pJAK2 or p-STAT6 proteins relative to total JAK or total STAT6 was calculated.
10
Protein Expression Analysis of HXR9 and CXR9 Treatment
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Cells were treated with 60 μmol/L HXR9 or CXR9 for 2 hours. Total proteins were extracted by using RIPA lysis buffer with protease inhibitor cocktail and separated by SDS‐PAGE, blotted onto PVDF, then immunoreacted with primary antibody overnight at 4°C. The primary antibodies used were anti‐PBX (sc‐28313 at 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti‐HOXB7 (ab51237 at 1:50; Abcam, Cambridge, MA, USA), anti‐HOXC6 (ab151575 at 1:1000; Abcam), anti‐HOXC8 (ab86236 at 1:1000; Abcam), anti‐Caspase‐3 (#9662 at 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti‐poly ADP ribose polymerase (anti‐PARP, #5625 at 1:1000; Cell Signaling Technology), anti‐c‐FOS (sc‐447 at 1:200; Santa Cruz Biotechnology), anti‐PI3K (#4249 at 1:1000; Cell Signaling Technology), anti‐AKT (#9272 at 1:1000; Cell Signaling Technology), anti‐p‐AKT (#5012 at 1:1000; Cell Signaling Technology), anti‐signal transducer and activator of transcription‐6 (anti‐STAT6, #5397 at 1:1000; Cell Signaling Technology), anti‐p‐STAT6 (#9364 at 1:1000; Cell Signaling Technology) and anti‐GAPDH (ZS‐25778 at 1:2000; ZSGB‐BIO, Beijing, China). Goat antirabbit IgG (ZB‐2301 at 1:5000; ZSGB‐BIO) and goat antimouse IgG (ZB‐2305 at 1:5000; ZSGB‐BIO) were used as the secondary antibodies.
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