Protein extracts were quantified by DC Protein assay (Bio-Rad), and equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis. After transfer of the proteins onto a nitrocellulose membrane (GE10600001, Sigma) for 1 hr at 100 mV, membranes were blocked in 5% milk, and specific proteins were labeled with the following antibodies: CPEB4 (Abcam Ab83009/clone 149C/D10, monoclonal homemade); HuR (3A2, sc-5261 Santa Cruz); HIF1a (Cayman 10006421); phospho-p44/42 (Erk1/2) (Thr202/Try204) (Cell Signaling clone E10, 9106); SOCS1 (Abcam Ab9870); phospho-p38 (Thr180/Y182) (Cell Signaling, 9211S); p38α (C-20)-G (Santa Cruz, sc-535-G); phospho-MAPKAPK2 (Thr222) (Cell Signaling, 3044S), TTP (D1I3T) (Cell Signaling, 71632), and vinculin (Abcam Ab18058). Quantification was done with ImageStudioLite software, and protein expression was normalized by the loading control signal.
Socs1
Manufactured by Abcam
Sourced in United States
SOCS1 is a protein that plays a role in the regulation of cytokine signaling pathways. It functions as a negative regulator of cytokine signaling by inhibiting the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
P stat3, by Abcam (3 mentions)
Socs3, by Abcam (3 mentions)
Gapdh, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
β actin, by Abcam (2 mentions)
Stat1, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ge10600001, by Merck Group (1 mentions)
Cpeb4, by Abcam (1 mentions)
Hur 3a2, by Santa Cruz Biotechnology (1 mentions)
Hif1a, by Cayman Chemical (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
N n n n tetramethylethylenediamine temed, by Beyotime (1 mentions)
P jak2, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Hif 1α, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
16 protocols using socs1
1
Western Blot Analysis of Protein Expression
2
Sceptridium ternatum Herba Biomarker Evaluation
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Sceptridium ternatum Herba was collected from Lishui, Zhejiang, China. Voucher specimens were identified by Prof. Xilin Chen of Zhejiang Chinese Medicine University. A voucher specimen of Sceptridium ternatum was deposited in the herbarium of the College of Pharmacy, Zhejiang Chinese Medical University (Hangzhou, China; 310053).
β-actin was purchased from Sigma Co., Ltd. (St. Louis, MO, USA). HIF1α, STAT3, p-STAT3, JAK2, p-JAK2, and SOCS1 were obtained from Abcam Co., Ltd. (Cambridge, MA, USA). Horseradish peroxidase-labeled Goat anti-Rabbit IgG and Goat anti-Mouse IgG were purchased from Beijing Zhongshan Jinqiao Co., Ltd. (Beijing, China). For western blot, 30% acrylamide/Bis (29:1), ammonium sulfate, twelve sodium dodecyl sulfate, N,N,N',N'-tetramethylethyle nediamine (TEMED), and western blot membrane washing solution were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). For real-time quantitative PCR (RT-qPCR), primers were obtained from Shanghai Sangon Biological Technology Service Co., Ltd. (Shanghai, China). The RNA extraction reagent TriZol was purchased from Bio RT Reagent Kit Co., Ltd. (Vancouver, Canada). PrimeScriptTM (Perfect Real Time) was supplied by Bao Biotechnology Co., Ltd. (Dalian, China).
β-actin was purchased from Sigma Co., Ltd. (St. Louis, MO, USA). HIF1α, STAT3, p-STAT3, JAK2, p-JAK2, and SOCS1 were obtained from Abcam Co., Ltd. (Cambridge, MA, USA). Horseradish peroxidase-labeled Goat anti-Rabbit IgG and Goat anti-Mouse IgG were purchased from Beijing Zhongshan Jinqiao Co., Ltd. (Beijing, China). For western blot, 30% acrylamide/Bis (29:1), ammonium sulfate, twelve sodium dodecyl sulfate, N,N,N',N'-tetramethylethyle nediamine (TEMED), and western blot membrane washing solution were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). For real-time quantitative PCR (RT-qPCR), primers were obtained from Shanghai Sangon Biological Technology Service Co., Ltd. (Shanghai, China). The RNA extraction reagent TriZol was purchased from Bio RT Reagent Kit Co., Ltd. (Vancouver, Canada). PrimeScriptTM (Perfect Real Time) was supplied by Bao Biotechnology Co., Ltd. (Dalian, China).
3
Western Blot Analysis of Liver Proteins
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Proteins were extracted from liver tissues and cell lysates, and the concentration was measured through a BCA Protein Assay Kit (Thermo Fisher Scientific, Shanghai, China). The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted onto polyvinylidene fluoride membranes (Millipore, USA). These membranes were blocked in nonfat dry milk (5% w/v) with Tris-buffered saline containing 0.1% Tween 20 (TBS-T) at 4 °C overnight and then incubated with primary antibodies against GAPDH (Cell Signaling Technology, Danvers, MA, USA), VDR, suppressor of cytokine signaling (SOCS) 1, and p62 (Abcam, Shanghai, China). After three washes with TBS-T, the membranes were incubated with peroxidase-conjugated secondary antibody (Cell Signaling Technology, Shanghai, China) for 1 h at room temperature. The densitometry of bands was quantified by using the Quantity One software for image analysis.
4
Protein Expression Analysis in Glomeruli
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Proteins isolated from MMCs and isolated glomeruli samples were separated by SDS–PAGE and transferred to nitrocellulose (NC) membranes, blocked with 5% nonfat dried milk and incubated at 4°C overnight with primary antibodies, including MHC class II (Abcam, Cambridge, MA, USA), SOCS1 (Abcam), β-actin (Abcam), phosphorylated STAT1 (P-STAT1) and STAT1 antibodies (Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies were subsequently applied, and the signals were detected using the ChemiDoc-It 600 Imaging System (UVP, Upland, USA). Densitometry analysis was performed using an ImageJ analysis system. All experiments were repeated at least three times.
5
Signaling Protein Expression Analysis
6
Western Blot Analysis of Brain Proteins
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Total proteins from brain tissues or cultured cells were lysed in RIPA buffer (Cell Signaling Technology, MA, USA) with 1 mM phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor (MCE, NJ, USA). BCA protein assay kit (Generay Biotechnology, Shanghai, China) was used to quantified protein concentrations. Equal amount of protein was applied for SDS-PAGE electrophoresis and then transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked with 5% skim milk at room temperature for 1 h, then incubated with primary antibodies C3 (1:1000, Abcam), iNOS (1:1000, Abcam), S100A10 (1:1000, Abcam), Arg1 (1:1000, Cell Signaling Technology), suppressor of cytokine signaling 1 (SOCS1) (1:1000, Abcam) and β-actin (1:5000, Cell Signaling Technology) overnight at 4 °C. HRP-conjugated secondary antibodies were used for further incubation with the membranes for 120 min, and signals on blots were developed by the ECL reagents (Millipore, MA, USA) and were analyzed by ImageJ software.
7
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.
8
Neutrophil Protein Isolation and Western Blot Analysis
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Protein isolation from cultured neutrophil was performed as previously described [16 (link)]. Western blots were performed using the standard SDS-polyacrylamide gel electrophoresis method and enhanced chemiluminescence detection reagents (GE Healthcare Biosciences AB, Uppsala, Sweden). Antibodies against phosphorylated signal transducer and activator of transcription 1 (pSTAT1) (Multi-clonal, Abcam, Cambridge, USA), STAT1 (Multi-clonal, Cell signaling technology, Beverly, MA), SOCS1 (Multi-clonal, Abcam, Cambridge, USA), and GAPDH (Cell signaling technology, Beverly, MA) were used. Immunoreactivity was semi-quantitatively measured by gel densitometric scanning and analyzed by the MCID image analysis system (Imaging Research, Inc.).
9
Western Blot Analysis of Key Signaling Proteins
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Cells were lysed in RIPA buffer (Beyotime, China). The protein was separated in 10% SDS-PAGE gel and transferred to nitrocellulose membranes. The 5% non-fat milk was applied to the membrane to block non-specific antigens. After incubation with primary and secondary antibodies (Beyotime Biotech.), the protein content was detected using an ECL kit (Pierce, USA). Primary antibodies used in this study were as follows: UBE2D3 (Abcam, Ab176568); STAT3 (Abcam, Ab68153); p-STAT3 (Abcam, Ab76315); HK2 (Abcam, Ab209847); PFKL (Abcam, Ab181064); PTP1B (Abcam, Ab244207); SHP-1 (Abcam, Ab32559); SHP-2 (Abcam, Ab32159); SOCS1 (Abcam, Ab62584); SOCS3 (Abcam, Ab16030); Ubiquitin (Abcam, Ab7780); β-actin (Abcam, Ab8227).
10
Protein Expression Analysis in Cell Samples
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Samples of cells and cortical tissues were lysed in cold RIPA lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with protease inhibitor cocktail and the protein concentration was determined using a BCA protein assay kit (Nanjing KeyGen Biotech Co., Ltd.). Proteins (20-40 µg) were subjected to 10% SDS-PAGE (Thermo Fisher Scientific, Inc.) and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 5% non-fat milk for 2 h at room temperature. The primary antibodies used were anti-Alix (1:500; cat. no. sc-53540; Santa Cruz Biotechnology, Inc.), anti-CD63 (1:1,000; product code ab213090; Abcam), anti-suppressor of cytokine signaling-1 (SOCS-1; 1:1,000; product code ab62584; Abcam), and anti-β-actin (1:3,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) and the membranes were incubated with the primary antibodies for 12 h at 4°C. Goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:3,000; product nos. 7076 and 7074, respectively; Cell Signaling Technology, Inc.) were used for detection for 2 h at room temperature. The signals were then detected using an enhanced chemiluminescent kit (GE Healthcare; Cytiva). Finally, ImageJ software (v1.8.0; National Institutes of Health) was used for densitometry.
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