The treated BMDMs were fixed with 4% paraformaldehyde for 10 min and blocked with blocking buffer (10% goat serum and 0.05% Triton X-100 in PBS) for 30 min. The cells were then incubated with indicated primary antibody (dilution 1:200) for 24 hours at 4°C and Cy3-conjugated secondary antibody (dilution 1:400) at room temperature for 2 hours. The positively stained cells were visualized under fluorescence microscope with 200 × magnification. Primary antibodies included anti-total SHP-1, anti-p-SHP-1, p-STAT6 (Signal transducer and activator of transcription 6) and acetyl-STAT3 (Abcam, Cambridge, MA and Cell signaling technology, Danvers, MA), anti-mouse TNF-alpha and IL-6 (R&D systems, Minneapolis, MN). The positively stained cells were quantitatively analyzed on Image J software and data was presented as ratio of arbitrary units to controls.
P stat6
Manufactured by Abcam
Sourced in United Kingdom, United States
P-STAT6 is a recombinant protein that represents the phosphorylated form of the signal transducer and activator of transcription 6 (STAT6) protein. STAT6 is a key transcription factor involved in various cellular processes. The phosphorylated form, P-STAT6, plays a role in the signaling pathways regulated by STAT6.
Automatically generated - may contain errors
Lab products found in correlation
Stat6, by Abcam (4 mentions)
P stat3, by Abcam (3 mentions)
P stat1, by Abcam (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
P stat1, by Cell Signaling Technology (2 mentions)
P jak2, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti p shp 1, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Anti cd206 pe, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (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)
Total protein extraction kit, by Beyotime (1 mentions)
14 protocols using p stat6
1
Immunostaining of Macrophage Signaling Proteins
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
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.
5
Apigenin Modulates Allergic Responses
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Apigenin (purity ≥95%), OVA (grade V), aluminum hydroxide, and histamine
dihydrochloride were purchased from Sigma-Aldrich Co, St Louis, Missouri.
Montelukast was obtained from Cipla Limited, Mumbai, India. Mouse OVA-specific
IgE, total IgE, total IgG1, β-hexosaminidase, IL-4, IL-5, IL-13, IL-17, IFN-γ,
and Leukotriene C4 enzyme-linked immunosorbent assay (ELISA) kit were obtained
from Bethyl Laboratories Inc, Montgomery, Texas. The primary antibodies of
GATA3, T-bet, NF-κB, IκBα, p-STAT6, suppressor of cytokine signaling 1 (SOCS1),
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Abcam,
Cambridge, Massachusetts. Total RNA extraction kit and real time-polymerase
chain reaction (RT-PCR) kit were purchased from MP Biomedicals India Private
Limited, India.
dihydrochloride were purchased from Sigma-Aldrich Co, St Louis, Missouri.
Montelukast was obtained from Cipla Limited, Mumbai, India. Mouse OVA-specific
IgE, total IgE, total IgG1, β-hexosaminidase, IL-4, IL-5, IL-13, IL-17, IFN-γ,
and Leukotriene C4 enzyme-linked immunosorbent assay (ELISA) kit were obtained
from Bethyl Laboratories Inc, Montgomery, Texas. The primary antibodies of
GATA3, T-bet, NF-κB, IκBα, p-STAT6, suppressor of cytokine signaling 1 (SOCS1),
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Abcam,
Cambridge, Massachusetts. Total RNA extraction kit and real time-polymerase
chain reaction (RT-PCR) kit were purchased from MP Biomedicals India Private
Limited, India.
6
Ephedra Aconite Asarum Decoction Modulates STAT6
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IMDCs in culture were plated into 6-well plate at 2 × 107 cells/ml, 2 ml per well, set 3 experimental groups including group C, group L, and group M. Groups L and M were induced with LPS (1 μg/ml, Sigma), and group M was also treated with Ephedra Aconite Asarum Decoction (2 mg/ml), harvest cells after 24 hours. Clearly, group L was the model control group. Total protein was extracted from each group and expressions of STAT6 (Proteintech) and pSTAT6 (phospho Y641, Abcam) were detected in each group.
7
Tissue Microarray Analysis of JAK-STAT Pathway
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Tissue microarrays were performed using the SIDSCO-TMA70 system (Scientific Integration Design Service Corp., Kaohsiung, Taiwan). Two tissue blocks were prepared for each case. In addition to hematoxylin and eosin staining, the sections were immunostained with the following primary antibodies: p-JAK2 (1:100, ab108596, Abcam, Cambridge, UK), p-STAT1 (1:200, #9167s, Cell Signaling Technology, Danvers, MA, USA), p-STAT3 (1:200, #9145s, Cell Signaling Technology), p-STAT5 (1:400, #9314s, Cell Signaling Technology), and p-STAT6 (1:100, ab28829, Abcam, Cambridge, UK). Sections were then incubated with goat anti-rabbit HRP. Finally, all slides were mounted with xylene-based mounting medium and scanned at 400× using the MoticEasyScan Pro (Motic in Asia, Xiamen, China). The quantitative intensity of staining was analyzed using Image-Pro Plus software (Version 6.0, Media Cybernetics, LP, USA). The immunohistochemistry intensity was determined as follows: staining percentage ≤25% positive cells as weak staining (-); 26–50% positive cells as mild to moderate staining (+/++); and >65% positive cells as intense staining (+++).
8
Quantitative STAT3/STAT6 Protein Analysis
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Western blot analysis was implemented as previously mentioned [27 (link)]. The primary antibodies used in this experiment are as follows: total and phosphorylated STAT3 (S727) (p-STAT3) (Abcam); total and phosphorylated STAT6 (Y641) (p-STAT6) (Abcam); PPAR-γ (Abcam); IL4Rα (Signalway Antibody); and β-actin (Signalway Antibody). The secondary antibody was goat anti-rabbit IgG (HRP) (Signalway Antibody). The blots were analyzed using ImageJ software.
9
Immunoblotting Antibody Protocol
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The following antibodies were purchased from Cell Signalling Technology: pSTAT1 (#8826), Arginase‐1 (#9819), Rab7 (#9367), Rab5 (#2143), BIP (#3183), EEA1 (#2411), IκBα (#9242), LAMP1 (#3243), Na+/K+ ATPase 1 (#3010), Lamin A/C (#2032), TAK1 (#4505), JNK (#9258), p‐JNK (#9251), p‐c‐Jun (#9165) and Vimentin (#5741). Antibodies purchased from Abcam were as follows: pSTAT6 (ab54461), CD36 (ab133625), CD14 (ab182032) and MSR1 (ab151707, ab79940). Antibodies against K63‐specific ubiquitin (#05‐1308) and ITGAM (PAB12135) were from Millipore and Abnova, respectively. Sheep antibodies against MSR1, TAK1, TAB1, TAB2, MKK7 and MKK4, and rabbit IgG were generated by the Antibody Production Team of the Division of Signal Transduction Therapy (DSTT), Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, United Kingdom. The antibody used for MSR1 immunohistochemistry was clone SRA‐E5 (from Abnova, #MAB1710). Commercial antibodies were used according to the manufacturer instructions. DSTT‐made antibodies were used at 2 μg/ml in TBS‐T containing 5% non‐fat‐dried milk. Recombinant proteins, plasmids and antibodies generated for the present study are available to request on our reagents website (https://mrcppureagents.dundee.ac.uk/ ).
10
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.
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