Media were removed and the cells were washed twice in ice-cold phosphate-buffered saline (PBS), scraped and collected as pellets after centrifugation at 1700 g for 5 min. The pelleted cells were incubated in RIPA buffer with proteinase and phosphatase inhibitors for 15 min. Lysates were then collected and centrifuged at 208,000 × g for 15 min at 4°C. Protein concentrations were measured using the DC Protein Assay Kit (Biorad, Cat. No. 5000111). SDS–PAGE and immunoblotting were performed as described previously in pre-cast bis-Tris 4–20% gradient gels (Invitrogen)64 (link). The following antibodies were used: IL2rγ (Abcam ab180698, bioss bs-2545R); IL13ra1 (Abcam ab79277); pAKT-S473 (CST 9271); Stat1 (CST 9172); Stat3 (CST 4904); pStat3-S727 (CST 9134); pStat2-Y690 (4441); pStat5-Y705 (CST 9145); Stat6 (abcam ab28829); Stat6 (CST); pStat6-Y641 (CST 56554); pStat5-Y694 (CST 4322); pStat5 (CST 9359); Jak1 (CST 3344); pJak1-Tyr1034/1035 (CST 74129); Jak2 (CST 3230T); Jak3 (CST 8827); pJak3 (CST 5031); Hexokinase II (CST 2867); Enolase (Abcam ab155102); pERK-p44/42 (CST 4370); cMyc (CST 5605); Pim3 (Abcam ab71321); and β-Actin (Sigma-Aldrich, A2228).
Stat6
Manufactured by Abcam
Sourced in United States
STAT6 is a protein that functions as a transcription factor, playing a role in the regulation of gene expression. It is involved in the signaling pathways of various cytokines and is important for the differentiation of T cells.
Automatically generated - may contain errors
Lab products found in correlation
P stat6, by Abcam (4 mentions)
Stat1, by Abcam (3 mentions)
P stat1, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
β actin, by Abcam (2 mentions)
Phospho stat6, by Cell Signaling Technology (2 mentions)
Stat4, by Cell Signaling Technology (2 mentions)
Stat2, by Abcam (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (1 mentions)
P stat3, by Abcam (1 mentions)
Total protein extraction kit, by Beyotime (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Minibest universal rna extraction kit, by Takara Bio (1 mentions)
Recombinant human interleukin 4 il 4, by Thermo Fisher Scientific (1 mentions)
Ppar r, by Abcam (1 mentions)
Jagged1, by Abcam (1 mentions)
Cleaved notch1, by Abcam (1 mentions)
10 protocols using stat6
1
Comprehensive Protein Profiling Technique
2
Proteomic analysis of S1P signaling
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Tissue or cellular proteins were extracted and subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio‐Rad, Hercules, CA). Rabbit anti‐S1P, S1PR3, phosphorylated signal transducer and activator of transcription (p‐STAT) 1, p‐STAT3, p‐STAT6, STAT1, STAT3, and STAT6 (Abcam) and β‐actin (Cell Signaling Technology, Danvers, MA) were used.
3
Dendrobium officinale Immune Regulation Protocol
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Dendrobium officinale was purchased from Zhejiang Shouxiangu Pharmaceutical Co., Ltd. (Jinhua, China), and PMA and LPS were purchased from Sigma (Saint Louis, MI, USA). Recombinant human interleukin-4 (IL-4) was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). TNF-α and IL-6 ELISA kits were purchased from Lianke Biological Technology (Hangzhou, China). Cell Counting Kit-8 (CCK-8) Assay Kit was received from MCE (Romulus, NJ, USA). Total Protein Extraction kit was purchased from Beyotime (Shanghai, China). The MiNiBEST Universal RNA Extraction Kit, PrimeScript RT Reagent kit, and SYBR Premix Ex Taq II Kit were purchased from TaKaRa (Dalian, China). Transwell polycarbonate membrane had an 8 μm pore size (Corning City, NY, USA). The primary antibodies E-cadherin, N-cadherin, Vimentin, Caspase-3, Bax, Bcl-2, Ki67, and β-actin were purchased from Proteintech (Wuhan, China). The primary antibodies ARG1, TGM2, and Cleaved-NOTCH1 were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies STAT6, p-STAT6, PPAR-r, JAGGED1, Cleaved-NOTCH1, and NOTCH1 were purchased from Abcam (Cambridge, UK). β-actin was purchased from Proteintech (Wuhan, China). The ECL Plus Western Blotting Detection Kit was purchased from Technology Co., Ltd. (Beijing, China). Anti-CD80-FITC and Anti-CD206-PE were obtained from Thermo Fisher (Waltham, MA, USA).
4
Immunoblotting Analysis of Signaling Pathways
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Whole protein extracted from RAW264.7 macrophages were resolved by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels according to standard procedures. The samples were then transferred to polyvinylidene difluoride membranes (Bio-Rad) and incubated at 4 °C overnight with antibodies against phosphorylated IKK-α/β (1:500, Cell Signaling Technology), IKK-α (1:1000, Cell Signaling Technology), IKK-β (1:1000, Cell Signaling Technology), phosphorylated NF-κBp65 (1:500, Cell Signaling Technology), NF-κBp65 (1:1000, Cell Signaling Technology), phosphorylated I-κBα (1:1000, Cell Signaling Technology), I-κBα (1:1000, Cell Signaling Technology), PPAR-γ (1:500, Cell Signaling Technology), phosphorylated STAT6 (1:500, Abcam), STAT6 (1:1000, Abcam) and the endogenous control GAPDH (1:10000, KangChen Bio-tech). The blots were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000, KangChen Bio-tech) at room temperature for 1 h. Immunoreactive bands were detected with ECL Western blotting kit (Thermo Scientific Pierce) and exposed to films and developed. The density of the immunoblots was measured by Image J (National Institutes of Health) and normalized by GAPDH.
5
Immunohistochemical Analysis of Lung Macrophages
6
Western Blot Analysis of Macrophage Markers
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Cells or exosomes were collected and lysed in Laemmli buffer (Sigma, USA). The protein concentration was determined with BCA Protein Assay kit (Beyotime). Protein lysates were separated by SDS-PAGE and transferred onto NC filter membrane (Millipore). Then the NC filter membrane was subsequently incubated with the primary antibody: iNOS (Abcam), Arg1 (Abcam), p-STAT1 (Abcam), p-STAT6 (Abcam), STAT1 (Abcam), STAT6 (Abcam), SOCS2 (Abcam), PKM2 (Cell Signaling Technology, CST, Danvers, MA, USA), HIF-1α (Invitrogen, Life Technologies, Carlsbad, CA, USA), β-actin (Abcam). β-actin was used as a control. After incubation with secondary antibodies and ECL (enhanced chemiluminescence, Millipore), the protein bands were captured by Bio-Rad ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). Quantitative analysis of protein bands was conducted with ImageJ software.
7
Protein and Signaling Pathway Analysis
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Protein extraction and western blot assays were conducted as previously described39 (link). The primary antibodies against α-SMA (ab7817, 1:300 dilution), collagen I (ab138492, 1:1000 dilution), GAPDH (ab8245, 1:5000 dilution), Smad3 (ab40854, 1:1000 dilution) and phospho-Smad3 (Ser 423 and Ser 425, ab52903, 1:1000 dilution) were purchased from Abcam (MA, USA); STAT6 (#9362, 1:1000 dilution), phospho-STAT6 (Tyr641, #9361, 1:1000 dilution), Akt (#9272, 1:1000 dilution), phospho-Akt (Ser473, #9271, 1:1000 dilution), ERK 1/2 (p44/42 MAPK, #4695, 1:1000 dilution) and phospho-Erk1/2 (Thr202/Tyr204, #4370, 1:1000 dilution) from Cell Signaling Technology (MA, USA) and IL4Rα (YT2337, 1:1000 dilution), TGFβ RI (YT4627, 1:1000 dilution) and TGFβ RII (YT4628, 1:1000 dilution) from ImmunoWay (TX, USA).
8
Western Blot Analysis of Exosomal Proteins
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RIPA (Genepharma) was used for extracting total protein. BCA was used for total protein quantification. SDS-PAGE (10%) was used to separate the proteins. The proteins were then transferred onto PVDF membranes (MilliporeSigma). Primary antibodies targeted against CD63 (cat. no. ab134045, 1:1,000; Abcam, Cambridge, MA, USA), TSG101 (cat. no. ab125011, 1:1,000; Abcam), p-STAT6 (cat. no. ab263947, 1:1,000; Abcam), STAT6 (cat. no. ab32108, 1:1,000; Abcam), N-cadherin (cat. no. ab76011, 1:1,000; Abcam), E-cadherin (cat. no. ab40772, 1:1,000; Abcam), calnexin (cat. no. ab133615, 1:1,000; Abcam), PTEN (cat. no. ab267787, 1:1,000; Abcam), Akt (cat. no. ab8805, 1:1,000; Abcam), p-Akt (cat. no. ab38449, 1:1,000; Abcam) and β-actin (cat. no. ab8226, 1:1,000; Abcam) were used to incubate the membranes at 4°C overnight after blocking for 1 h with skimmed milk (5%). HRP-conjugated secondary antibodies (cat. no. ab288151, 1:5,000; Abcam) were used to incubate the membranes. The protein bands were visualized using ECL (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). IPP 6.0 (Image-Pro Plus 6.0) was applied for densitometric analysis.
9
Immunostaining of STAT Proteins in Lung Development
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Immunostaining was performed on paraformaldehyde-fixed and paraffin-embedded excised lungs and embryos of different gestational ages (15.5 -21.5 dpc) as previously described [26] [27] [28] . Primary antibodies for STAT1 (1: 200, Abcam Inc., UK), STAT2 (1: 50, Abcam Inc.), STAT3 (1: 50, Cell Signaling Technology Inc., USA), STAT4 (1: 50, Cell Signaling Technology Inc.), STAT5 (1: 50, Santa Cruz Biotechnology Inc., USA), and STAT6 (1: 50, Abcam Inc.) were used. Negative control reactions included omission of the primary antibody and the simultaneous omission of the primary and secondary antibodies. In both the cases, immunoreactive staining was not observed. Sections were incubated with a labeled streptavidin-biotin immunoenzymatic antigen detection system (UltraVision Large Volume Detection System Anti-Polyvalent, Horseradish Peroxidase, Lab Vision Corporation, USA) according to the manufacturer's instructions. At least three independent experiments were performed for each antibody tested. In each experiment, a different set of slides (duplicates included, two sections from the same individual, per slide) comprising the whole range of gestational ages plus the adult were used. Different and unrepeated animal samples were selected for each group (gestational age). Six different animals for each group per studied antibody were examined.
10
Quantifying STAT Protein Levels
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Pooled tissue samples (n=3 subjects from the same litter, per sample) of both experimental groups at the selected gestational ages were processed for Western blot analysis. Proteins were obtained according to Kling et al [29] , and the protocol was performed as previously described [28] . Blots were probed with antibodies to STAT1 (1: 2000, Cell Signaling Technology Inc.), STAT2 (1: 2000, Abcam Inc.), STAT3 (1: 2000, Cell Signaling Technology Inc.), STAT4 (1: 1000, Cell Signaling Technology Inc.), STAT5 (1: 2000, Cell Signaling Technology Inc.), STAT6 (1: 2000, Abcam Inc.), phospho-STAT3 (1: 1000, Cell Signaling Technology Inc.), phospho-STAT6 (1: 1000, Cell Signaling Technology Inc.), and SOCS3 (1µg/mL; Abcam Inc.) according to the manufacturer's instructions. For loading control, blots were probed with β-tubulin (1: 150000, Abcam Inc.), and the quantitative analysis was performed with Quantity One 4.6.5 1-D Analysis Software (Bio-Rad). For analysis of each antibody, at least three independent experiments were performed (n=3) in each experiment an unrepeated pooled sample was used. In total, nine animals were used in each group (gestational age/condition) per antibody.
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