Western blotting was performed as described in previous publications 76 (link)–78 (link). Antibodies used were rabbit anti-human FOXM1 (1:1000, 5436; Cell Signaling Technology), rabbit anti-human cyclin B1 (1:1000, 12231, Cell Signaling Technology), and rabbit anti-human β-actin (1:2000, 4970; Cell Signaling Technology). Resultant immunological bands were observed using the ChemiDoc XRS Plus System (Bio-Rad Laboratories). The intensity of the bands was quantified using ImageJ software (National Institutes of Health, Bethesda, MD). Uncropped, full-length original gel images are shown in Supplementary Figs. S1 and S2 .
Rabbit anti human β actin
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-human β-actin is a primary antibody that specifically binds to the β-actin protein found in human cells. It is a commonly used control and normalization reagent in various cell biology and biochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated secondary antibody, by GE Healthcare (3 mentions)
Ecl reagent, by Cytiva (3 mentions)
Tbs t, by GE Healthcare (2 mentions)
Tbs t, by Merck Group (2 mentions)
Primary rabbit anti human ido1, by Merck Group (2 mentions)
Perfecta sybr green fastmix, by Quanta Biosciences (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Chemidoc xrs plus system, by Bio-Rad (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Anti eif4e, by BD (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Secondary horseradish peroxide coupled goat anti rabbit igg h l, by Fortis Life Sciences (1 mentions)
1 methyl dl tryptophan, by Merck Group (1 mentions)
Recombinant human bfgf, by R&D Systems (1 mentions)
18 protocols using rabbit anti human β actin
1
Western Blotting Protein Detection
2
Western Blot Analysis of ERβ Expression
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For Western blot cells were lyzed in RIPA buffer containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and total protein concentration was measured using Pierce™BCA protein assay kit (Pierce Biotechnology, Rockford, USA). Equal amounts of protein, 100 μg, were separated using SDS/Page on mini-protean TGX gels (BioRad Laboratories, USA) and transferred to PVDF membranes (BioRad Laboratories, USA), which were incubated with mouse anti-human ERβ (Leica Biosystems, Newcastle, United Kingdom), rabbit anti-human β-actin (Cell Signaling Technology, Danvers, MA, United States) and horseradish peroxidase (HRP)- conjugated secondary antibodies from Dako (Dako, Glostrup, Denmark). Proteins were visualized using ECL detection reagent (Ge Healthcare, Buckinghamshire, UK) and ChemiDoc™Touch Imaging System and Image lab 5.2.1 analysis software from BioRad. A Western blot of ERβ expression of all cell lines and a negative control is shown in Supplementary Figure 3 .
For quantification of ERβ an ELISA kit, Cat# E90437Hu (Wuhan USCN, TX, USA), was used.
For quantification of ERβ an ELISA kit, Cat# E90437Hu (Wuhan USCN, TX, USA), was used.
3
Quantifying Platelet Protein Levels
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Platelets lysed in Laemmli buffer were separated by SDS PAGE gel electrophoresis and probed for eIF-4E, vinculin and β-actin. After protein transfer to a nitrocellulose membrane, membranes were blocked in 5% non-fat milk in Tris-buffered saline with Tween-20 (TBS-T, Sigma-Aldrich, Taufkirchen, Germany) for 1 h. Afterwards, primary monoclonal antibodies were added. eIF4E, actin and vinculin were detected using specific mouse anti-human antibodies (1:1000) (anti-eIF4E, BD Transduction Laboratories, San Diego, CA, USA; anti-actin, ICN Biomedicals, Costa Mesa, CA, USA; anti-vinculin, Upstate Biotechnology, Placid, NY, USA) and incubated overnight at 4 °C with constant agitation. Membranes were washed in TBS-T repeatedly, and HRP-conjugated secondary antibody (1:10,000) (GE Healthcare, Freiburg, Germany) was added for 1 h at room temperature. After washing, chemiluminescent substrate (ECL reagent, Amersham Biosciences, Munich, Germany) was added for 1–5 min and bands were visualized on plain film. β-Actin served as loading control (1:1000, rabbit anti-human β-Actin, Cell Signaling Technology, Dallas, TX, USA).
4
Quantifying IDO Expression and Activity
5
Quantifying HSP27 and Phospho-HSP27 in Myocardial Infarction
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Platelets lysed in Laemmli buffer were separated by SDS PAGE gel electrophoresis and probed for HSP27, phospho-HSP27, and β-actin. After protein transfer to a nitrocellulose membrane, membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween-20 (TBS-T, Sigma-Aldrich, Taufkirchen, Germany) for 1 h. Afterwards, primary monoclonal antibody to HSP27 or phospho-HSP27 (1:1000, Santa Cruz Biotechnologies, Dallas, TX, USA) were added and incubated overnight at 4 °C with constant agitation. Membranes were washed in TBS-T repeatedly, and HRP-conjugated secondary antibody (1:10000) (GE Healthcare, Freiburg, Germany) was added for 1 h at room temperature. After washing, chemiluminescent substrate (ECL reagent, Amersham Biosciences, Munich, Germany) was added for 1–5 min and bands were visualized on plain film. β-Actin served as loading control (1:000, rabbit anti-human β-Actin, Cell Signaling Technology, Dallas, TX, USA). HSP27 and phospho-HSP27 levels were quantified by band densitometry analysis with the help of HSP27- and pHSP27-to-actin ratios. Mean HSP27- and pHSP27-to-actin ratios for the myocardial infarction group were set as 1 and relative increases in HSP27 and pHSP27 levels in the control group were compared accordingly. Relative HSP27 and pHSP27 levels between groups based on actin loading are shown.
6
Investigating Transcriptional Regulation in Cells
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All tissue culture components and solutions were purchased from Gibco BRL (Paisley, UK). Recombinant human bFGF and recombinant human HB-EGF were purchased from R&D Systems (Abingdon, UK). AG1478, LY294002, PD98059, and SP600125 were obtained from Calbiochem (Bad Soden, Germany). SB203580 was from Tocris (Ellisville, MO). Human-specific small interfering RNA (siRNA) against nuclear factor of activated T cell 5 (NFAT5) and nontargeted control siRNA were obtained from Qiagen (Hilden, Germany). All other agents used were from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise. The following antibodies were used: a neutralizing goat anti-bFGF (R&D Systems), a neutralizing goat anti-HB-EGF (R&D Systems), a neutralizing rabbit anti-transforming growth factor (TGF)-β (pan specific; R&D Systems), a rabbit anti-human NFAT5 (1:200; Santa Cruz Biotechnology, Dallas TX), a rabbit anti-human β-actin (1:1000; Cell Signaling, Frankfurt/M., Germany), and anti-rabbit IgG conjugated with alkaline phosphatase (1:2000; Cell Signaling).
7
Quantification of Kindlin-3 in Platelets
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Platelets (1 × 109) lysed in Laemmli buffer were separated by SDS PAGE and stained for kindlin-3. After protein transfer to a nitrocellulose membrane, membranes were blocked in 5% nonfat milk in TBS-T for 1 h. Afterward, primary antibody to full-length kindlin-3 (1:100 in 3% nonfat milk/TBS-T, Abcam, Cambridge, UK) was added and incubated overnight at 4 °C with constant agitation. Membranes were washed in TBS-T repeatedly and HRP-conjugated secondary antibody (1:10,000, GE Healthcare, Freiburg, Germany) was added for 1 h at room temperature. After washing, chemiluminescent substrate (ECL reagent, Amersham Biosciences, Munich, Germany) was added for 1–5 min and bands were visualized on plain film. β-Actin served as loading control (rabbit anti-human β-Actin, Cell Signaling Technology, Dallas, TX, USA). Kindlin-3 protein levels were quantified by band densitometry analysis with the help of a kindlin-3 to actin ratio.
8
Regulation of Autophagy and Apoptosis in Glucose Deprivation
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Sugar-free DMEM medium (Glucose concentration 0mmol/L, GIBCO, USA), DMEM medium (glucose concentration 25mmol/L, HyClone USA), annexin V-FITC Apoptosis Detection Kit (Beijing 4A Biotech Co., Ltd), CCK8 Kit (Wuhan Boster Biological Technology Co., Ltd), siRNA Lyophilized Powder (Shanghai GenePharma Co., Ltd), mRFP-GFP-LC3 adenovirus (Han Heng Biotechnology Co., Ltd), rabbit anti-human β-actin, LC3AB, Cleaved Caspase 3, FoxO1 and Rab7 monoclonal antibody (Cell Signaling Technology, USA), Beclin-1 Rabbit anti-human monoclonal antibody, P62 mouse anti-human monoclonal antibody, Caspase 3 rabbit anti-human monoclonal antibody (Abcam, USA), rabbit anti-human SIRT1, Ac-FoxO1 polyclonal antibody (Santa Cruz, USA), horseradish peroxidase-labeled goat anti-mouse secondary antibodies, goat anti-rabbit secondary antibodies(Pierce, USA).
9
Quantifying HIF1-α and VEGF Expression
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Western blot analysis of HIF1-α and VEGF: Skin flap tissues were homogenized in Laemmli sample buffer and determined using the bicinchoninic acid protein assay for protein content. Then, 20 μg of total protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore) by electroblotting. After washing with tris-buffered saline solution in 0.1% Tween-20 (Merck) (TBS-T), the members were blocked in 5% w/v fat-free milk powder (Roth) for 1 hour at room temperature and then washed with TBST. The members were incubated with primary antibody: HIF1-α (Cell Signaling), VEGF (Cell Signaling), and rabbit antihuman β-actin (Cell Signaling) overnight at 4°C. After washing, the members were incubated with an HRP-labeled secondary antibody (goat anti-rabbit IgG; Cell Signaling) for 1 hour at room temperature. Bound antibodies’ images were developed by ECL reagent (ECL Advance Western Blotting Detection Kit, GE Healthcare) in the dark and detected using a fluorescence imaging analysis system. The protein expression was quantified by ImageJ software (NIH).
10
Quantifying GMF-β Protein Levels
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Western blotting was carried out routinely. The primary antibodies included rabbit anti-human GMF-β (NBP1–89755, dilution 1:200; Novus Biologicals, Littleton, CO, USA) and rabbit anti-human β-actin (#5125, dilution 1:1000; Cell Signaling, Boston, MA, USA). Protein bands were visualized with enhanced chemiluminescence (Amersham, Piscataway, MA, USA) following manufacture's instruction. GMF-β expression level was normalized to β-actin expression for each sample.
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