Cells were pretreated with 10 µM GANT61 for 4 hours and then treated with 20 ng/ml M-Csf with or without 20 ng/mL Il-4 for 1 hr in combination with GANT61 or DMSO and/or SHH (100 nM). After one hour cells were processed using the Simple Chip Plus Enzymatic kit (Cell Signaling) as per manufacturer’s protocol. 10 µg of cross-linked chromatin was immunoprecipitated with either 2 µg of anti-Stat6 (Cell Signaling) or anti-Gli1 (Novus). Chromatin was eluted from the IP and cross-links were reversed followed by column purification of DNA. Purified DNA from ChIP and input was subjected to real-time quantitative PCR to quantitate the amount of DNA associated with Gli or Stat6 in the Il4ra or Il4 promoter sequence. PCR was done using 2X Maxima SYBR Green Master Mix (Thermo Scientific) with primer pairs to amplify various regions of the respective promoters. Primer pairs and associated schematic are detailed in Supplementary Figure 9. CT values of input DNA was used to calculate percent input of immunoprecipitation utilizing the following calculation: Percent Input = 2% x 2(C[T] 2% Input Sample-C[T] IP Sample) and percent enrichment as compared to corresponding controls is depicted. Each reaction was done in triplicate using an Applied Biosystems Step One Plus.
Anti stat6
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-STAT6 is a primary antibody that detects STAT6 (Signal Transducer and Activator of Transcription 6), a transcription factor involved in the IL-4 and IL-13 signaling pathways. It can be used for applications such as Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti p stat6, by Cell Signaling Technology (9 mentions)
Anti phospho stat6, by Cell Signaling Technology (6 mentions)
Anti akt, by Cell Signaling Technology (5 mentions)
Anti stat1, by Cell Signaling Technology (5 mentions)
Anti jnk, by Cell Signaling Technology (4 mentions)
Anti stat3, by Cell Signaling Technology (4 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Anti erk1 2, by Cell Signaling Technology (3 mentions)
Anti phospho stat1, by Cell Signaling Technology (3 mentions)
Anti p akt, by Cell Signaling Technology (3 mentions)
Anti pparγ, by Cell Signaling Technology (3 mentions)
As1517499, by Axon Medchem (3 mentions)
Anti nf κb p65, by Cell Signaling Technology (3 mentions)
Anti jak1, by Cell Signaling Technology (3 mentions)
Anti p38, by Cell Signaling Technology (2 mentions)
Anti phospho jnk, by Cell Signaling Technology (2 mentions)
Anti phospho p38, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
30 protocols using anti stat6
1
Chromatin Immunoprecipitation Assay for Stat6 and Gli1
2
Protein Quantification and Validation Methodology
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Aliquots of cell lysate (50 µg/lane) were separated by electrophoresis on 10% or 12% SDS polyacrylamide gels. Anti-phospho-STAT6, anti-STAT6, anti-phospho-SMAD2 (#3104), anti-SMAD2, anti-phospho-SMAD3, anti-SMAD3, anti-phospho-SMAD1, 5, and 9, anti-SMAD1, 5, and 9, anti-phospho-ERK1/2 (44/42 MAPK) (#4370), anti-ERK1/2 (#9102), anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, and anti-SDHA (#5893) antibodies were purchased from Cell Signaling Technology (Beverley, MA, USA). Anti-NDUFA9 and COX-4 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-UQCRC2 (ab14745) was purchased from Abcam (Cambridge, UK). Anti-ATP5A1 was purchased from Invitrogen (Thermo Fisher). Secondary antibodies (goat anti-mouse and goat anti-rabbit) were obtained from Santa Cruz Biotechnology. Images were scanned on an ODYSSEY instrument and quantified using Image Studio Digits (LI-COR Biosciences, Lincoln, NE, USA). Full length uncropped blots are presented in Supplementary Figures 12 –17 .
3
Comprehensive Immunoblotting Profiling of Cellular Signaling
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Five-7 x 106 cells were prepared by lysis in a buffer made up of 20 mM Tris, 150 mM NaCl, 2 mM EDTA (ethylenediaminetetraacetic acid), 2 mM EGTA (ethylene glycol-tetra-acetic acid), 0.5% v/v Triton X-100 supplemented with protease inhibitor cocktail (Sigma), 1 mM DTT (dithiothreitol; Amersham Biosciences), 1 mM PMSF (phenyl-methyl-sulfonyl fluoride; Sigma), 1 mM okadaic acid (Sigma) and phosphatase inhibitor cocktail (Thermo Scientific). Proteins were subjected to SDS-PAGE, transferred to PVDF membranes and immunoblotted with the following primary Abs: anti-CK2α (provided by Dr. M. Ruzzene, University of Padova, Italy), anti-CK2β, anti-RELA, anti-FOXO1 (Abcam), anti-pRELA S529 (recognizes S527 in mouse), anti-IRF4, anti-BLIMP-1, anti-BCL6 (Santa Cruz), anti-GAPDH (Ambion), anti-pAKT S129 (provided by Dr. M. Ruzzene, University of Padova, Italy), anti-AID (Invitrogen), anti-NOTCH2 (D76A6), anti-pAKT S473, anti-AKT, anti-pERK1/2 T202/Y204, anti-pBTK Y223, anti-ERK1/2, anti-pPTEN S380/T382/T383, PTEN, anti-pFOXO1 S256, anti-pSTAT6 Y705, anti-STAT6 (Cell Signaling). Secondary Abs: anti-rabbit IgG HRP-linked Ab (Cell Signaling), HRP labeled goat anti-mouse IgG (KPL), goat anti-rat IgG HRP-conjugated (Calbiochem), donkey anti-goat IgG HRP-conjugated (Santa Cruz).
4
Interleukin-13 Receptor Alpha-1 Signaling Pathway
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Proteins were extracted from the hearts of Il13ra1−/− and WT mice or H9C2 cells using a RIPA buffer (Sigma‐Aldrich, St. Louis, MO) supplemented with Complete Mini, EDTA‐free, protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, catalog number: 11 836 170 001). Following separation on an SDS‐PAGE, proteins were transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen Corporation, Carlsbad, CA). Membranes were stained with a primary antibody overnight at 4°C, washed, and incubated with the appropriate secondary antibody for 45 to 60 minutes at room temperature. Specific reactive bands were detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL). The antibodies used were anti–signal transducer and activator of the transcription (STAT)3 (Cell Signaling Technology, Beverly, MA, catalog number 9139), anti‐STAT6 (Cell Signaling Technology, Beverly, MA, catalog number 5397), anti–phosphorylated STAT3 (Cell Signaling Technology, Beverly, MA, catalog number 9145), anti–phosphorylated STAT6 (Santa Cruz Biotechnology, Dallas, TX, catalog number sc‐11762‐R), anti‐actin (Santa Cruz Biotechnology, Dallas, TX, catalog number sc‐58670), and anti–α tubulin (Sigma‐Aldrich, Saint Louis, MO, catalog number T9026).
5
STAT6 Activation Detection by Western Blot
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Cells were lysed in RIPA extraction solution (15 mM Tris, pH 7.5, 120 mM NaCl, 25 mM KCl, 2 mM EGTA, 2 mM EDTA, 0.1 mM dithiothreitol, 0.5% Triton X-100, and protease inhibitor cocktail [Sigma]). Protein concentration was assessed by BCA assay. Total protein lysates (25 μg/lane) were subjected to SDS-PAGE and transferred onto an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore Corp.). Antibodies used were: anti STAT6 (Cell Signaling Technology); anti phospho-STAT6 (Cell Signaling Technology) and anti-β-actin antibody as loading control (Abcam). Western blots were quantified by Quantity One Analysis software (Bio-Rad).
6
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.
7
Multiplex Immunoblotting Antibody Validation
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All chemicals were obtained from Sigma-Aldrich unless otherwise specified. The following antibodies were used in this study: rabbit anti-UCP1 (Abcam, ab155117, GR3233606-10 1:2000), anti-Parvalbumin (ABclonal, A13538, Lot0054370201 1:1000), anti-Transferrin (Abbkine, ABM40235, 1:1000), anti-pSTST6 (Cell Signaling Tech, 56554, 1:2000), anti-STAT6 (Cell Signaling Tech, 9362, 1:2000), anti-pAKT (Ser473) (Cell Signaling Tech, 4060, 1:2000), anti-pAKT (Thr308) (Cell Signaling Tech, 13038,1:2000), anti-AKT (Cell Signaling Tech, 2920, 1:2000), anti-GAPDH (Santa Cruz, sc32233, 1:1000), anti-GFP (Proteintech, 50430-2-AP, 1:1000), anti-pERK (Cell Signaling Tech, 4370, 1:2000), anti-ERK (Cell Signaling Tech, 4695, 1:2000), anti-pPKC (Abcam, ab180848, 1:2000), anti-PKC (Abcam, ab179522, 1:2000), anti-p4EBP1 (Cell Signaling Tech, 2855, 1:2000), anti-4EBP1 (Cell Signaling Tech, 9452, 1:2000), anti-ACTB (Sigma Aldrich, A3854, 1:10000), anti-Flag (Sigma-Aldrich, F7425, 1:10000), HRP-conjugated goat anti-Rabbit IgG (Cell Signaling Tech, 7074, 1:10000), anti-pGSK-3β (Cell Signaling Tech, 5558, 1:2000), anti-GSK-3β (Cell Signaling Tech, 12456, 1:2000). Rictor antibody (Abcam, ab70374, 1:100), mTOR antibody (Cell Signaling Tech, 2983, 1:100) (Supplementary Table 2 ).
8
Signaling Pathway Antibodies and Assays
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Anti–phosphorylation (p) inhibitor of B (IκB)-α, anti-PKG, anti-VASP, and anti–p-VASP (Ser 239), anti-Arginase1, anti-STAT6, anti–p-STAT6 (Tyr 641), and β-actin antibodies were from Cell Signaling Technology (Beverly, MA). Anti-eNOS mouse polyclonal antibody was obtained from BD Biosciences (Lexington, KY). Anti-GAPDH rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Biosource total Akt and p-Akt (Ser 473) ELISA kits were purchased from Invitrogen (Carlsbad, CA), and IRS2 and p-IRS2 ELISA kits were purchased from Cell Signaling Technology. Lactate was measured using an Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen). (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-IM1,2-diolate (DETA-NO) was purchased from Enzo Life Sciences. DETA-NO was used within 24 h after the reconstitution.
9
Western Blot Analysis of STAT1 and STAT6
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The cells were washed with phosphate-buffered saline (PBS) and lysed with cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM NaF, and Complete Protease Inhibitor Cocktail; Roche, Indianapolis, IN, USA) on ice for 30 min. Thirty to fifty micrograms of the cell lysate was resolved by SDS-PAGE on 10% or 12% gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies followed by peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Calbiochem, EMD Chemicals Inc., San Diego, CA, USA) and the ECL reagent (Millipore) for band visualization. To verify equal loading and adequate transfer, the membranes were probed with anti-α -GAPDH antibodies (Santa Cruz Biotechnology). The primary antibodies were anti-STAT1, anti-phospho-STAT1, anti-STAT6, and anti-phospho-STAT6 (Cell Signaling Technology, Beverly, MA, USA).
10
Immunoblotting of B Cell Signaling
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B cells cultured under various conditions were harvested and cell lysates were prepared in lysis buffer. The lysate was resolved on a 10% reducing SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. After blocking with Tris-buffered saline (pH 7.4) containing 5% dried skimmed milk, the membrane was incubated with antibodies, followed by probing with HRP-conjugated anti-rabbit or -mouse antibody (Sigma). The bands were detected by chemiluminescence with ECL detection reagents (Life Technologies). Antibody to Phospho-STAT6 (Tyr641, 56,554), anti-STAT6 (5,397), anti-p-PKA C(4,781), anti-PKA C-α (5,842), anti-p-CREB (9,198), anti-CREB (9,197), PI3 Kinase p110 δ 34,050, anti-p-Akt (Ser473, 4,060), anti-Akt (4,685), anti-p-FoxO1 (Ser256, 9,461), anti-FoxO1 (2,880), anti-p-FoxO3a (Ser253, 13,129), anti-FoxO3a (12,829), anti-PPARγ (2,443) were from Cell Signaling Technology (Danvers, MA, USA). Anti-ubiquitin (PTM-1106) was from PTM BIO (Beijing, China). Anti-β-Actin (66009-1-Ig) was from Proteintech.
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