Cell lysates were collected by digesting with RIPA buffer (Beyotime, Nanjing, China) and protease inhibitors (Sigma-Aldrich, USA). BCA kit determined protein concentration (Thermo Fisher, USA). Thirty µg protein were loaded on 10% or 15% SDS-PAGE gels and transferred onto nitrocellulose membranes. The membranes were incubated with specific first antibodies and corresponding second antibody. Primary antibodies purchased from Abcam (Cambridge, UK) included anti-E-cadherin (ab182733, 1:1,000), anti-N-cadeherin (ab13847, 1:500), anti-Vimentin (ab44976, 1:2,000), anti-JAK2 (ab108596, 1:5,000), anti-p-JAK2 (ab32101, 1:5,000), anti-STAT3 (ab31369, 1:1,000), anti-p-STAT3 (ab30645, 1:1,000), anti-β-catenin (ab6302, 1:4,000) and anti-β-actin (ab8245, 1:1,000). Secondary antibodies included goat anti-mouse IgG (ab6789, 1:5,000; Abcam) and goat anti-rabbit IgG (ab6721, 1:5,000; Abcam).
Ab30645
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab30645 is a laboratory equipment product. It is designed for use in scientific research and analysis. The core function of this product is to [Description not available].
Automatically generated - may contain errors
Lab products found in correlation
Ab16030, by Abcam (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Ab203883, by Thermo Fisher Scientific (3 mentions)
Anti ifnγ 500 p119, by Thermo Fisher Scientific (3 mentions)
Ab3987, by Abcam (3 mentions)
Ab11524, by Abcam (3 mentions)
Ab28829, by Abcam (3 mentions)
Ab32520, by Abcam (3 mentions)
Ab64693, by Abcam (3 mentions)
Ab15323, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Ab13847, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab108596, by Abcam (2 mentions)
Ab31369, by Abcam (2 mentions)
Ab32101, by Abcam (2 mentions)
Ab2071, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
11 protocols using ab30645
1
Western Blot Analysis of EMT and JAK/STAT Markers
2
Immunoblotting Assay for Macrophage Markers
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Immunoblotting was performed as previously described [48 (link)] using the following primary antibodies: anti-iNOS (ab15323, Abcam), anti-CD16 (ab203883), anti-IFNγ (500-P119, PeproTech, Rocky Hill, NJ, USA), anti-Arg1 (sc-271430, Santa Cruz), anti-CD206 (ab64693, Abcam), anti-IL-4 (ab11524, Abcam), anti-STAT1 (ab3987, Abcam), anti-p-STAT1 (ab30645, Abcam), anti-STAT6 (ab32520, Abcam), anti-p-STAT6 (ab28829, Abcam), anti-SOCS3 (ab16030, Abcam), and the HRP-conjugated secondary antibody (1:5000). The plots were visualized by ECL Plus (Thermo).
3
Western Blot Analysis of Signaling Proteins
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Cell lysates were prepared with RIPA buffer containing protease inhibitors (Sigma). Protein concentrations were measured with the BCA Protein Assay according to the manufacturer’s manual (Beyotime Institute of Biotechnology). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Membranes were incubated overnight at 4 °C with a 1:1000 solution of antibodies (Cell Signaling Technology). A secondary antibody was then used for immunostaining for one hour at room temperature. The primary antibody used here are anti-EGFR antibody (Abcam, ab52894, 1:1000, Cambridge, MA), anti-STAT1 antibody (Abcam, ab30645, 1:1000) and anti-Bcl-2 antibody (Abcam, ab32124, 1:1000).
4
Western Blot Analysis of Protein Markers
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Whole cell lysates were prepared as previously described.18 (link) Equal amounts of protein were boiled, separated on 10% SDS-PAGE, transferred onto a PVDF membrane, and visualized using an enhanced chemiluminescence kit (Millipore, MA, USA). Antibodies against PTPRO (ab150834), STAT3 (Y705) (ab76315), STAT3 (S727) (ab30647), STAT3 (ab68153), STAT2 (Tyr690) (#88410; Cell Signaling), STAT2 (ab134192), STAT1 (Y701) (ab30645), STAT1 (ab31369), JAK2 (Y1007+Y1008) (ab32101), JAK2 (ab108596), c-MYC (s62) (ab51156), c-MYC (ab39688), human PD-L1 (ab205921), mouse PD-L1 (ab233482), and β-actin (ab6276) were purchased from Abcam. All uncropped western-blot figures are presented in online supplementary figures s13‒16 .
5
Immunostaining of Phosphorylated STAT Proteins
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Immunostaining was performed on sections (2 μm) of formalin-fixed, paraffin-embedded ERC-derived tissue. All slides were processed on a BenchMark Ultra automated immunostainer (Ventana Medical Systems, Tucson, AZ) according to the manufacturer's instructions. The following primary antibodies were used: anti-phosphorylated STAT1 (ab30645, Abcam, Cambridge, UK) and anti-phosphorylated STAT3 (ab76315, Abcam). Positive reactions were visualized through oxidation of diaminobenzidine resulting in brown staining. Finally, sections were counterstained with hematoxylin, and image acquisition was performed with a Leica microscope. Immune cells and epithelial cells were identified by morphology. Nontumor-bearing tissue from 6 hepatic resections of hepatocellular carcinoma served as controls. All histopathology specimens were reviewed by 3 expert pathologists.
6
Western Blot Analysis of Inflammatory Markers
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sfd-FLSs were homogenized using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Protein concentration was measured using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Subsequently, protein samples (20 µg in each lane) were separated by 12.5% SDS-PAGE. Protein were blotted on a nitrocellulose membrane and the membranes were incubated with primary antibodies anti-IL-17 (1:1,000; ab193955; Abcam), TNF-α (1:1,000; ab109332; Abcam), ERK (1:1,000; ab32537; Abcam), pERK (1:1,000; ab201015; Abcam), STAT1 (1:1,000; ab2071; Abcam), pSTAT1 (1:1,000; ab30645; Abcam) and β-actin (1:1,000; ab8226; Abcam) for 12 h at 4°C, after blocking with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at 37°C. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. PV-6001; OriGene Technologies, Inc.) for 24 h at 4°C. The blots were visualized using an enhanced chemiluminescence detection system (cat. no. 32209; Pierce; Thermo Fisher Scientific, Inc.). Densitometric quantification was performed using Quantity-One software (version 1.2; Bio-Rad Laboratories, Inc.).
7
Molecular Mechanisms of miR-155 Inhibitor on BMSCs
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BMSCs transfected with miR-NC inhibitor or miR-155 inhibitor were seeded in 6-well plates (1×104 cells/well) and cultured with or without 50 mM puer-arin for 144 h. BMSCs were lysed using RIPA buffer (P0013B, Beyotime Institute of Biotechnology) and centrifuged at 12,000 × g for 10 min at 4°C. The protein concentration was determined by a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Protein (40 μg per lane) was loaded on 12% SDS-PAGE and subsequently transferred to PVDF membranes (Abcam) followed by blocking with 5% BSA (Sigma-Aldrich; Merck KGaA) and incubation with primary antibodies [anti-caspase-3 (1:1,000, ab13847), anti-caspase-9 (1:1,000, ab202068), anti-VEGF (1:1,000, ab53465), anti-p53 (1:1,000, ab131442), anti-TNF-α (1:1,000, ab6671), anti-STAT1 (1:1,000, ab2071, Abcam), anti-pSTAT1 (1:1,000, ab30645), anti-BMP-2 (1:1,000, ab214821), anti-macrophage colony-stimulating factor (M-CSF, 1:1,000, ab52846) and anti-β-actin (1:1,000, ab8226); all from Abcam] for 12 h at 4°C. After washing with PBST (0.5% Tween-20), the proteins were incubated with goat anti-rabbit IgG antibody (1:5,000, ab6721, Abcam) at room temperature for 2 h. All bands were visualized with an ECL system kit (MultiSciences). Band densities were analyzed by ImageJ software, version 4.6 (National Institutes of Health).
8
Western Blot Analysis of Signaling Proteins
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Cells were lysed using RIPA lysis buffer containing protein kinase and phosphatase inhibitors for 30 minutes on ice. Tissue samples were homogenized by sonication and proteins were extracted. Protein concentrations were determined using a BCA kit (Thermo Fisher). Samples (20 μg) were separated by SDS‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocked in 5% skim milk for 1 hour, the membranes were probed with primary antibodies towards RANKL (SC‐7627, Santa Cruz Biotechnology), Pros1 (SC‐271326, Santa Cruz Biotechnology), Tyro3 (SC‐271326, Santa Cruz Biotechnology), SOCS1 (ab9870, Abcam), SOCS3 (ab16030, Abcam), STAT1 (ab31369, Abcam), phospho (p)‐STAT1 (phosphor Y701, ab30645, Abcam), STAT3 (cat. no. 12640, Cell Signaling) and p‐STAT3 (Tyr705, cat. no. 07‐2173, Millipore), respectively, at 4°C overnight. The membranes were subsequently incubated with a horseradish peroxidase (HRP)‐conjugated secondary antibody at room temperature for 2 hours, and the protein bands were visualized using enhanced chemiluminescence. All data were normalized to ‐actin.
9
Immunoblotting for Macrophage Markers
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Immunoblotting was performed as previous describe [48] using the following primary antibodies: anti-iNOS (ab15323, Abcam,), anti-CD16 (ab203883)), anti-IFNγ (500-P119, PeproTech, Rocky Hill, NJ, USA), anti-Arg1 (sc-271430, santa cruz), anti-CD206 (ab64693, abcam), anti-IL-4 (ab11524, Abcam), anti-STAT1 (ab3987, Abcam), anti-p-STAT1 (ab30645, Abcam), anti-STAT6 (ab32520, Abcam), anti-p-STAT6 (ab28829, Abcam), anti-SOCS3 (ab16030, Abcam), and the HRP-conjugated secondary antibody (1:5000). The plots were visualized by ECL Plus (Thermo).
10
Quantification of Protein Phosphorylation
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Cells were cleaned for three times in phosphate buffer solution (PBS), and the total protein was separated by radioimmunoprecipitation assay (RIPA) buffer (Beyotime). Bicinchoninic acid (BCA) protein assay kit (CoWin Biotechnology) was applied to detect protein concentration. Total proteins were electrophoresed to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA, USA) treated with 5% non-fat milk for 1 h. Protein were identified overnight at 4°C by specific primary antibodies: p-JAK2 (ab195055, 1:1000; Abcam), JAK2 (ab39636, 1:5000; Abcam), p-STAT1 (ab125685, 1:3000; Abcam), STAT1 (ab30645, 1:1200; Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab9485, 1:1,200; Abcam). Then the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (ab205718, 1:2,000; Abcam), and the bands on membranes were visualized by the enhanced chemiluminescence (ECL) reagent (Beyotime).
The analyzed samples were normalized by β-actin.
The analyzed samples were normalized by β-actin.
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