After transfection for 48 h, the total cellular protein was extracted using the radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China), and the protein concentrations were measured using the bicinchoninic acid protein assay kit (BCA) (Beyotime, Shanghai, China). The boiled protein samples were electrophoresed on 10% SDS-polyacrylamide gels, and then transferred onto a PVDF membrane (Beyotime, Shanghai, China). The membrane containing protein fractions was blocked with QuickBlockTM Blocking Buffer (Beyotime, Shanghai, China) for 30 min and incubated with primary antibodies for 12 h, at 4 °C, including FZD7; β-Catenin (1:2000, Huabio, Hangzhou, Zhejiang, China, 0407-16); BAX (1:2000, Proteintech Group, Chicago, IL, USA, 50599-2-Ig); Bcl2 (1:1000, Bioss, Beijing, China, bsm-33047M); Caspace-3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, 14220); and β-actin (1:5000, Proteintech Group, Chicago, IL, USA, 66009-1-Ig). After washing, the membrane was incubated with secondary antibodies for 2 h, at 26 °C. Protein bands were visualized using an ECL advanced western blotting detection kit (Beyotime, Shanghai, China). β-actin served as the loading control.
β catenin
Manufactured by Huabio
Sourced in China
β-catenin is a protein that plays a key role in the Wnt signaling pathway, which is involved in various cellular processes, including cell-cell adhesion, cell fate determination, and embryonic development. It functions as a transcriptional co-activator and regulates the expression of genes involved in these processes.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Abcam (2 mentions)
Ecl advanced western blotting detection kit, by Beyotime (1 mentions)
Bcl 2, by Bioss Antibodies (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
β actin, by Proteintech (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Quickblocktm blocking buffer, by Beyotime (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
β actin, by Huabio (1 mentions)
Runx2, by Huabio (1 mentions)
Image pro plus 6, by Bio-Rad (1 mentions)
Stat3, by Huabio (1 mentions)
Total protein extraction kit, by Signalway Antibody (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Nucleoprotein extraction kit, by Sangon (1 mentions)
Rankl, by Abcam (1 mentions)
Ubiquitin, by Proteintech (1 mentions)
E cadherin, by Proteintech (1 mentions)
7 protocols using β catenin
1
Protein Expression Analysis after Transfection
2
Protein Expression Analysis of Stem Cell Markers
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Rabbit anti-human CD133, β-catenin, NANOG, OCT4 and SOX2 antibodies were purchased from Huabio (China) for western blot analysis. Rabbit anti-human phospho-RARα (Ser77), RARβ and phospho-β-catenin (Tyr489) antibodies were bought from Anbo Biotechnology (China). Rabbit anti-human Akt, phospho-Akt (Thr308) and tubulin antibodies were purchased from Beyotime Biotechnology (China), and rabbit anti-human PI3K was bought from Antibody Revolution (USA). Western blots were conducted according to standard procedures.
3
Western Blot Analysis of Protein Expression
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At the end of the iPTH treatment, the total protein was extracted using the Total Protein Extraction Kit (Signalway Antibody, USA), the Nuclear protein was extracted using a Nucleoprotein Extraction Kit (Sangon Biotech, China). Western blotting was conducted as previously described.62 (link) Briefly, the protein concentration was determined using a BCA Protein Assay Kit (Beyotime, China). After denaturation, the samples were loaded onto SDS-polyacrylamide gel electrophoresis gels for electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk or 5% BSA and incubated with primary antibodies against β-actin (Huabio, China), STAT3 (Huabio, China), phospho-STAT3 (Tyr705) (Cell Signaling Technology, USA), RUNX2 (Huabio, China), OSX (Abcam, UK), ALP (Huabio, China), OPN (Huabio, China), β-catenin (Huabio, China), RANKL (Abcam, UK) and OPG (Huabio, China) at 4 °C overnight, and rabbit and mouse secondary antibodies (Signalway Antibody, USA). The bands were visualized with a ChemiDoc Touch Imaging System (Bio-Rad, USA) and quantified with Image-Pro Plus 6.0.
4
Antibody and Chemical Reagents for Protein Analysis
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The following antibody was used in this study: VAMP8 (Proteintech, 15546–1-AP, 1:1000 for WB,1:500 for IHC), DDX5 (ZENBIO, R24094, 1:1000 for WB), DDX5 (Proteintech, 67025–1-Ig, 2 μg for IP), β-catenin (HUABIO, A6-F8, 1:2000 for WB), Vimentin (ZENBIO, R22775, 1:1000 for WB), E-cadherin (Proteintech, 20874–1-AP, 1:5000 for WB), N-cadherin (Proteintech, 22018–1-AP, 1:2000 for WB), ubiquitin (Proteintech, 10201–2-AP, 1:500 for WB), LC3B (Abmart, T55992, 1:1000 for WB), Flag (Proteintech, 66008–4-Ig, 1:5000 for WB), GAPDH (Affinity, AF7021, 1:10000 for WB).
The following chemicals were used in this study: Earle’s Balanced Salt Solution (Beyotime, C0213), Cycloheximide (MCE, HY-12320), Chloroquine (MCE, HY-17589A), MG132 (MCE, HY-13259).
The following chemicals were used in this study: Earle’s Balanced Salt Solution (Beyotime, C0213), Cycloheximide (MCE, HY-12320), Chloroquine (MCE, HY-17589A), MG132 (MCE, HY-13259).
5
Protein Expression Analysis in Cervical Cancer
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The total protein was extracted from C-33A and CaSki cells by RIPA buffer (Beyotime Institute of Biotechnology). The concentration of protein was detected by BCA kit (Beyotime Institute of Biotechnology). Subsequently, protein samples (20 µg/per lane) were separated with 10% SDS-PAGE and transferred onto PVDF membranes. Following blocking with non-fat milk at room temperature for 2 h, membranes were incubated with primary antibodies (all 1:1,000) as follows: OTX1 (cat. no. ab25985; Abcam), matrix metalloproteinase (MMP)2 (cat. no. ab181286; Abcam), MMP9 (cat. no. ab76003; Abcam), tissue inhibitor of MMP (TIMP)2 (cat. no. ab180630; Abcam), Wnt9 (cat. no. ER60346; Huabio), β-catenin (cat. no. ab223075, Abcam), adenomatous polyposis coli (APC; cat. no. ab239828; Abcam), Glycogen synthase kinase (GSK)-3β (cat. no. ab32391; Abcam) and Axis inhibition protein (AXIN)2 (cat. no. ab109307; Abcam) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated goat Anti-Rabbit IgG H&L secondary antibody (1:10,000; cat. no. ab205718, Abcam) for 1 h at room temperature. β-actin (1:1,000; cat. no. ab8226; Abcam) was used as an internal reference protein. The bands were visualized using a Novex™ ECL Chemiluminescent Substrate Reagent kit (Thermo Fisher Scientific, Inc.). The grey value was analyzed using ImageJ software (Version 1.45s; National Institutes of Health).
6
Protein Extraction and Analysis for Dental Cells
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DPCs were collected and lysed using RIPA buffer (KeyGEN, China) for 30 min at 4 °C. The nuclear protein was collected using cytoplasmic and nuclear extraction kit (Invent, USA). The protein lysates were quantified by BCA Protein Assay Kit (KeyGEN, China). Equal 15–20 μg protein extracts were loaded per lane. After primary antibody and PHRP-conjugated secondary antibody incubation, the protein bands were visualized using BLT GelView 6000Plus system (BLT, China). For pull-down assay, CDC42 activity was tested according to the CDC42 activation kit protocol (NewEastBiosciences, USA). Antibodies included CDC42 (1:1000, abcam), DMP1 (1:1000, Biovision), DSPP (1:1000, Zenbio), ALP (1:1000, Huabio), p-GSK3β (1:1000, Huabio), β-catenin (1:1000, Huabio), Col-1 (1:1000, abcam), GAPDH (1:5000, abcam), Occludin (1:1000, Abcam), E-cadherin (1:1000, Zenbio).
7
Extracellular Vesicle Protein Detection
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