The scaffolds were laid on the coverslips and placed in the 6-well plate. BMSCs and HUVECs were planted on the MBG-NH2, MBG-NH2/IGF and MBG-NH2/IGF@SF/VEGF scaffolds at a density of 5 × 104 cells/mL and cultured for 7 days. The washed cells were fixed with acetone and methanol (1:1). Then, 10% (v/v) horse serum was used for blocking. After being treated with the primary antibody against Bmp2 (Beyotime Biotech, Haimen, China, 1:200), Runx2 (Cell Signaling Technology, USA, 1:1600), CD31(Cell Signaling Technology, Danvers, MA, USA, 1:200) and β-actin (Beyotime Biotech, Haimen, China, 1:20) overnight, β-actin was labeled with FITC (1:100), while Bmp2, Runx2 and CD31 were labeled with Alexa Fluor 594 (1:100). Then 4′,6-diamidino-2-phenylindole (DAPI, Beyotime Biotech, Haimen, China) was used to stain the nucleus. Finally, 90% glycerol was used for sealing, and the sample was observed under CLSM.
β actin
Manufactured by Beyotime
Sourced in China, United States, United Kingdom
β-actin is a cytoskeletal protein that is widely expressed in eukaryotic cells. It is a component of the microfilament system and plays a crucial role in various cellular processes, including cell motility, structure, and division.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (42 mentions)
Bca protein assay kit, by Beyotime (34 mentions)
Ripa lysis buffer, by Beyotime (24 mentions)
Gapdh, by Beyotime (16 mentions)
Ripa buffer, by Beyotime (16 mentions)
Cleaved caspase 3, by Cell Signaling Technology (12 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (10 mentions)
Bcl 2, by Cell Signaling Technology (10 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (9 mentions)
Polyvinylidene difluoride membrane, by Merck Group (9 mentions)
Caspase 3, by Cell Signaling Technology (8 mentions)
Bca kit, by Beyotime (8 mentions)
Polyvinylidene fluoride membrane, by Merck Group (8 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (8 mentions)
Hrp conjugated secondary antibody, by Beyotime (8 mentions)
Caspase 3, by Abcam (7 mentions)
Bcl 2, by Proteintech (7 mentions)
Lysis buffer, by Beyotime (7 mentions)
Bca assay kit, by Beyotime (7 mentions)
Il 1β, by Abcam (7 mentions)
291 protocols using β actin
1
Scaffolding and Cell Culture Assay
2
Western Blot Analysis of Cellular Proteins
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Total proteins were extracted, and the concentration was detected by the bovine serum albumin method (Beyotime Institute of Biotechnology, P.R.C). For every sample, 40 μg of protein lysates underwent sodium dodecyl sulphate-polyacrylamide gel electrophoresis with 10% acrylamide gel used, which were then transferred onto polyvinylidene difluoride membranes (Millipore Corporation, USA) under 350 mA for 2.5 h. Non-fat milk (5%) was used to block the membrane, and primary antibodies against CELF1 (1:500, Abcam, USA), CDKN1B (1:1000, Sigma, USA), caspase-3 (1:1000, Abcam, USA), cleaved caspase-3 (1:1000, Abcam, USA) or β-actin (1:2000, Beyotime Institute of Biotechnology, P.R.C) were used for incubation overnight under 4 °C. Subsequently, horseradish peroxidase-conjugated secondary antibody (1:500, Beyotime Institute of Biotechnology, P.R.C) was used for incubation. After stripping, Western blot detection reagents of ultra-enhanced chemiluminescence (ECL, Beyotime Institute of Biotechnology, P.R.C) were utilised for reprobing the membrane with β-actin (1:2000, Beyotime Institute of Biotechnology, P.R.C). Protein was observed under an ECL system (ECL, Beyotime Institute of Biotechnology, P.R.C). Each Western band was quantified by densitometry, which was expressed in bar graphs.
3
Evaluation of CuSO4-Induced Cytotoxicity
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CuSO4, poly-vinylpyrrolidone, and C6H12O6 were obtained from Shanghai Tian Scientific Co. Ltd. Cell culture medium (Dulbecco's modified eagle’s medium, DMEM) were from Hyclone Laboratories. Fetal bovine serum (FBS) were got from Gibico. Acridine orange, ethidium bromide, FITC-Annexin-V/PI apoptosis assay kit, WST-1 assay kit and Hoechst 33342 were obtained from Beyotime Company of China. Antibody VEGFR2 (Flk-1 [C-1158], sc-504, rabbit polyclonal antibody raised against amino acids 1158–1345 of mouse Flk-1, Santa Cruz Biotechnology, USA). MMP-2 (H-76, sc-10736, rabbit monoclonal antibody against human MMP-2, Santa Cruz Biotechnology, USA). MMP-9 (H-129, sc-10737, rabbit monoclonal antibody against human MMP-9, Santa Cruz Biotechnology, USA). GAPDH (AG019, Primary antibodies used were mouse antibodies specific for the GAPDH, Beyotime Institute of Biotechnology, Jiangsu, China). F-actin (Mouse monoclonal [NH3] to F-actin, Abcam). β-actin (AA128, Primary antibodies used were mouse antibodies specific for the β-actin, Beyotime Institute of Biotechnology, Jiangsu, China). Secondary antibodies goat anti‑mouse (A0216, Beyotime Institute of Biotechnology, Jiangsu, China). Goat anti‑rabbit (A0208, 1:1000, Beyotime Institute of Biotechnology, Jiangsu, China).
4
Western Blot Analysis of ZEB2 in Osteosarcoma Cells
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A total of 25 μg proteins from the lysates of the osteosarcoma cells were added to electrophoresis through SDS/PAGE and then transferred them on to PVDF membranes. Then, these membranes were incubated with primary antibodies for ZEB2 (Abcam, Cambridge, MA, U.S.A.) or β-actin (Beyotime, Nantong, China) overnight at 4°C, and with horseradish peroxidase–conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) at room temperature for 2 h, respectively. Finally, the signals were detected by ECL Plus (Beyotime, Nantong, China) as per the manufacturer’s instructions and then calculated the relative protein levels with β-actin as the loading control.
5
Protein Extraction and Western Blot Analysis
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Total protein was extracted using Radio-Immunoprecipitation Assay (RIPA) buffer (Beyotime Co. Ltd). A bicinchoninic acid (BCA) kit (Beyotime Co. Ltd) was used to measure protein concentration. Proteins (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to polyvinylidene uoride membranes (PVDF; Millipore, Billerica, MA, USA). Antibodies used were RECK, Mortalin, E-cadherin, N-cadherin, p-STAT3 Tyr705 , Ac-STAT3 K685 and vimentin (Cell Signaling Technology, 1: 1000), GAPDH and β-actin (Beyotime Co. Ltd, 1: 500) (Supplementary Table S2).
Densitometric analysis was performed using Image-Pro-Plus 6.0 software, and GAPDH or β-actin served as the internal controls to correct for differences in protein loading.
Densitometric analysis was performed using Image-Pro-Plus 6.0 software, and GAPDH or β-actin served as the internal controls to correct for differences in protein loading.
6
Quantitative Western Blot Analysis of Uterine Proteins
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Proteins were extracted from uterine tissues at various stages after treatment, utilising ice-cold radioimmunoprecipitation assay (RIPA) buffer that was supplemented with protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Beyotime). Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime). Twenty μg protein samples were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% non-fat milk in TRIS-buffered saline at room temperature for 1 h, and then incubated in primary antibodies against KRT86 (Santa Cruz) and beta-actin (1:1000; Beyotime) at 4 °C overnight. After washing three times, the PVDF membranes were probed with secondary antibodies, 1:10,000 HRP-conjugated rabbit anti-goat or goat anti-mouse IgG (Zhongshan Biotechnology), for 1 h at room temperature. Finally, HRP-tagged bands were visualised using the Immobilon western chemiluminescent HRP substrate (WBKLS500, Milipore), and chemiluminescence intensity of each band was quantified by ImageJ software.
7
Western Blot Analysis of KOR in Mouse Brain
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Protein lysates of mouse brain tissue were prepared in RIPA:PMSF (100:1) supplemented with phosphatase inhibitors. Protein concentrations were measured by the bicinchoninic acid assay (Beyotime, Shanghai, China). Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Pall, East Hills, NY, United States). The PVDF membranes were blocked with 5% skim milk and incubated with primary antibodies reactive with KOR (1:1000; Abcam, Cambridge, MA, United States) or beta-actin (1:1000; Beyotime), at 4°C overnight. The blots were then incubated with horseradish peroxidase (HRP)–labeled secondary antibody (Proteintech, Shanghai, China) for 1 h. The membrane was incubated with SuperSignal West Pico Chemiluminescent Substrates (Thermo Fisher Scientific, Waltham, MA, United States) and bands visualized using an automatic multifunction chemiluminescent detection system (Tanon, Shanghai, China). The signals were calculated by densitometry using ImageJ software.
8
Western Blot Analysis of Stem Cell Markers
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The cultured SSCs were digested by RIPA (Beyotime, ShangHai, China) at 4 °C for 30 min and the protein were degenerated in 5×SDS sample loading buffer at 100°C for 10 min. Total protein was separated by SDS-PAGE 100V for 90 min, transferred to a 0.22-μm PVDF membrane at approximately 200 mA for 90 min, and incubated with B-ACTIN (1:1000, Beyotime, Shanghai, China), SOX9 (1:500, BOSTER, Wuhan, China), PCNA (1:1000, BOSTER), PLZF (1:300, Santa Cruz, USA), p-AKT (1:1000, Sangon Biotech, Beijing, China), AKT (1:1000, Sangon Biotech), ERK (1:1000, CST, Beverly, MA, USA), p-ERK (1:1000, CST). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse were used as a secondary antibody (1:2000, BOSTER). Detection was performed using the Thermo Scientific Pierce ECL western blot substrate (Thermo Scientific, USA). The results were analyzed with a Tanon-410 automatic gel imaging system (Tanon Corporation, Shanghai, China).
9
Protein Expression Analysis in SSCs
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Total cell extracts were prepared from SSCs cultured in control medium or media containing different concentration of PD0325901. Total proteins were resolved by PhosphoSafe (Millipore), and western blotting analysis was performed according to the protocol we described previously (Cao et al. 2011 (link), Yu et al. 2014) . Briefly, proteins were transferred to PVDF membrane and incubated with antibodies including b-actin (1:1000, Beyotime, Haimen, Jiangsu, China), PCNA (1:2000, Millipore), c-MYC (1:2500, Chemicon), ERK1/2 (1:1000, C.S.T), pERK1/2 (1:1000, C.S.T) at 4 8C for overnight. The next day, the secondary antibody were added, and incubated. Then, the detection was performed using the BM-chemiluminescence blotting substrate (Roche) (Yu et al. 2014 (link), Zhang et al. 2011) (link).
10
Protein Expression Analysis in Hepatic Samples
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The proteins were isolated from hepatic samples, and their concentrations were measured using BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). The lysate was mixed with 5× SDS sample buffer and boiled for 10 min. Lysate samples were separated on 6 % and 12 % SDS-polyacrylamide gels, and transferred to a PVDF membrane. The blots were blocked with 5 % milk blocking solution for 2 h at room temperature and then incubated overnight with antibodies against PER1 (1:1,000; Abcam, USA), mTOR (mammalian rapamycin), Phospho-mTOR (1:1,000; Cell Signaling Technology, USA), and β-actin (1:1,000; Beyotime Institute of Biotechnology). HRP-conjugated anti-rabbit IgG antibody (1:1,000; Beyotime Institute of Biotechnology) was used as the secondary antibody. The blots were visualized by ECL Western Blotting Detection Reagents (Beyotime Institute of Biotechnology) and the images were performed by GEL imaging system (Bio-Rad, USA). The quantification of proteins was analyzed by the software Quantity One (Bio-Rad, USA).
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