Western blot was performed as previously described (Ahn et al. 2019 (link)). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.
Rabbit anti β actin
Manufactured by Beyotime
Sourced in United States, United Kingdom
Rabbit anti-β-actin is a primary antibody used for the detection and quantification of the β-actin protein, a commonly used internal control and loading reference in various research applications. This antibody is produced in rabbits and specifically recognizes the β-actin isoform.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene uoride membrane, by Merck Group (2 mentions)
Horseradish peroxidase labeled secondary antibody, by Beyotime (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Horseradish peroxidase labelled secondary antibody, by Beyotime (1 mentions)
Ecl detection reagent, by Beyotime (1 mentions)
Rabbit anti collagen 1, by Abcam (1 mentions)
Rabbit anti psmad3, by Abcam (1 mentions)
Rabbit anti smad3, by Abcam (1 mentions)
Rabbit anti tgf β1, by Proteintech (1 mentions)
Rabbit anti lamin b1, by Abcam (1 mentions)
Rabbit anti c myc, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Beyotime (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence plus kit, by Beyotime (1 mentions)
Rabbit anti human vimentin, by Abcam (1 mentions)
Mouse anti human e cadherin, by Abcam (1 mentions)
Foxm1, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
7 protocols using rabbit anti β actin
1
Western Blot Analysis of Fibrosis Markers
2
Western Blot Analysis of Nuclear Proteins
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Tumor tissues or cells were washed with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Nantong, China) to obtain whole protein extracts. The nuclear protein lysates were prepared with a NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Proteins in the lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies: polyclonal rabbit antibody against human FOXM1 (1:1000; Abcam, Cambridge, UK), rabbit anti-Cyclin D1 (1:1000; Sigma-Aldrich Co., St Louis, MO, USA), rabbit antic-Myc (1:1000; Sigma-Aldrich Co.), rabbit anti-TCF-4 (1:500; Sigma-Aldrich Co.), rabbit anti-Lamin B1 (1:1000; Abcam), and rabbit anti-β-actin (1:1000; Beyotime Institute of Biotechnology). The signals from the primary antibody were amplified by incubating with horse radish peroxidase conjugated anti-rabbit IgG (1:1000; Beyotime Institute of Biotechnology) and detected with Amersham™ ECL™ Western Blotting Detection Reagents (GE Healthcare, Boston, MA, USA).
3
Western Blot Analysis of EMT Markers
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Whole protein extracts from SW620 at 72h following shRNA transfection or untransfection were lysed in ice-cold RIPA lysis buffer (Beyotime Inc., NanTong, China) according to manufacturer’s protocol. From each sample preparation 50μg of whole protein was separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). Standard Western blotting was performed using a polyclonal rabbit antibody against human FoxM1 (1:1000, Abcam, UK), mouse anti-human E-cadherin (1:1000, Abcam, UK), rabbit anti-human Vimentin (1:1000, Abcam, UK), rabbit anti-human Snail (1:500, Abcam, UK) and rabbit anti-β-actin (1:1000., Beyotime, china). The signals from the primary antibody was amplified by HRP conjugated anti-mouse IgG or anti-rabbit IgG (1:1000; Beyotime, china) and detected with Enhanced Chemiluminescence Plus kit (Beyotime, China).
4
Western Blot Analysis of NLRP3, GSDMD, Caspase-1
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The right brain cortex and retina protein samples (10 μg) were subjected to 8–12% SDS-PAGE and then transferred to PVDF membranes (Merk Millipore, Burlington, MA, USA). Blocked with 5% normal goat serum (NGS) for 2 h, the membranes were incubated with primary antibodies: rabbit Anti -NLRP3 (1:1000, Cat# ab263899; Abcam), rabbit anti-GSDMD (1:500, Cat# AF-4012; Affinity), rabbit anti-caspase-1 (1:1000, Cat# AF-4022; Affinity), and rabbit anti-β-actin (1:200; Cat# AF5003; Beyotime Biotechnology, Shanghai, China) at 4 °C for 24 h. The membranes were then incubated with anti-rabbit secondary antibodies (1:500; Cat# A0239; Beyotime Biotechnology, Shanghai, China) at 4 °C for 2 h. The protein signals were detected by Immobilon Western Chemiluminescent HRP substrate (Merk Millipore, Burlington, MA, USA) and analyzed using ImageJ analysis software (https://imagej.net/Fiji/Downloads , accessed on 30 May 2017, Bethesda, MD, USA).
5
Protein Extraction and Western Blotting
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Total proteins were extracted from different tissues using a Tissue or Cell Total Protein Extraction Kit (Sangon, Shanghai, China) and normalized with a BCA Protein Assay Kit (Sangon, Shanghai, China). Frozen tissues (50 mg per sample) were homogenized in lysis buffer (Beyotime, Shanghai, China) and centrifuged at 14000 rpm for 10 min at 4°C. Denatured proteins (40 mg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. After blocking with 5% skim milk in Tris-buffered saline containing Tween-20, the membranes were incubated with rabbit anti-MMP-9 (1:500 dilution, 25C19, Beyotime), rabbit anti-TGF-β1 (1:500 dilution, 27C10, Beyotime) or rabbit anti-β-actin (1:500 dilution, AG019, Beyotime). Horseradish peroxidase-conjugated goat anti-mouse IgG (Zymed, San Francisco, CA, USA) was used as the secondary antibody. Hybridized bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). The signals were quantified by densitometry. β-actin was used for as an internal protein control for normalization.
6
Quantitative Protein Analysis of Heart Tissue
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The proteins of heart tissue samples were loaded (50 µg) and subjected to 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene uoride membranes (EMD Millipore Corporation, Billerica, USA) for 1.5 h at 100 V. The membranes were blocked with 5% nonfat milk for 1 h and incubated with the primary antibodies: rabbit anti-SIRT1 (1:2,000, A nity Biosciences, OH, USA), rabbit anti-caspase-3(1:1,000, A nity Biosciences, OH, USA), rabbit anti-Bax (1:1,000, A nity Biosciences, OH, USA), rabbit anti-Bcl-2 (1:1,000, A nity Biosciences, OH, USA) or rabbit anti-β-Actin (1:1,500, Beyotime Institute of Biotechnology, Nantong, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labeled secondary antibody (1:5,000, Beyotime Institute of Biotechnology, Nantong, China). The bands were visualized using ECL plus enhanced chemiluminescence kit (A nity Biosciences, OH, USA). The intensity of protein bands was measured with the NIH Image J software (version 1.62; National Institutes of Health, Bethesda, MD, USA). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.
7
Quantitative Protein Analysis of Heart Tissue
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The proteins of heart tissue samples were loaded (50 µg) and subjected to 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene uoride membranes (EMD Millipore Corporation, Billerica, USA) for 1.5 h at 100 V. The membranes were blocked with 5% nonfat milk for 1 h and incubated with the primary antibodies: rabbit anti-SIRT1 (1:2,000, A nity Biosciences, OH, USA), rabbit anti-caspase-3(1:1,000, A nity Biosciences, OH, USA), rabbit anti-Bax (1:1,000, A nity Biosciences, OH, USA), rabbit anti-Bcl-2 (1:1,000, A nity Biosciences, OH, USA) or rabbit anti-β-Actin (1:1,500, Beyotime Institute of Biotechnology, Nantong, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labeled secondary antibody (1:5,000, Beyotime Institute of Biotechnology, Nantong, China). The bands were visualized using ECL plus enhanced chemiluminescence kit (A nity Biosciences, OH, USA). The intensity of protein bands was measured with the NIH Image J software (version 1.62; National Institutes of Health, Bethesda, MD, USA). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.
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