Total proteins were extracted from exosomes, whole-cell lysates, or homogenized femur tissue using the radioimmunoprecipitation assay lysis buffer (Beyotime, Haimen, China). Then western blot was performed according to a previous study (Guo et al. 2020 (link)). The primary antibodies against Alix, CD63, Calnexin, ALP, runt-related transcription factor 2 (Runx2), large tumor suppressor 1 (LATS1), and GAPDH were purchased from Abcam (1:1000). The primary antibodies against YAP, phosphor (p)-YAP, p-LAST1, TAZ, and p-TAZ were purchased from Cell Signaling Technology (1:1000). The signals were visualized using an ECL chemiluminescent system (Pierce, USA).
P taz
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
P-TAZ is a lab equipment product offered by Cell Signaling Technology. It functions as a tool for the detection and quantification of phosphorylated forms of the transcriptional activator TAZ (Transcriptional co-activator with PDZ-binding motif).
Automatically generated - may contain errors
Lab products found in correlation
P yap, by Cell Signaling Technology (3 mentions)
P last1, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Calnexin, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Calcein, by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Santa Cruz Biotechnology (1 mentions)
Cycloheximide (chx), by Cell Signaling Technology (1 mentions)
Ha ub, by Addgene (1 mentions)
Flag tag (flag), by Santa Cruz Biotechnology (1 mentions)
Flag taz, by Addgene (1 mentions)
Col10a1, by Thermo Fisher Scientific (1 mentions)
Edta free cocktail inhibitor tablets, by Thermo Fisher Scientific (1 mentions)
P lats1, by Cell Signaling Technology (1 mentions)
Lats1, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
6 protocols using p taz
1
Exosome Protein Profiling by Western Blot
2
Trp53, LAST1 and TAZ Regulation
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Antibodies against Trp53 (#2524; dilution 1:1000), LAST1 (#3477; dilution 1:1000), pLAST1 (#8654; dilution 1:1000), TAZ (#83669; dilution 1:1000), pTAZ (#59971; dilution 1:1000), HA (#5017; dilution 1:1000), GAPDH (#5174; dilution 1:1000); cycloheximide (CHX) and insulin-transferrin-sodium selenite media supplement (ITS Supplement) were purchased from Cell Signaling Technology. BrdU (#sc-32323; dilution 1:1000), β-TrCP (#sc-390629; dilution 1:1000), GFP (#sc-9996; dilution 1:1000) and flag (#sc-7945; dilution 1:1000) antibodies were obtained from Santa Cruz Biotechnology. Col10a1 (#14-9771-80; dilution 1:1000) antibody, EDTA-free cocktail inhibitor tablets, calcein and BrdU labeling were obtained from Fisher Scientific™. The Transfection Reagent FuGENE® HD was ordered from Promega (USA). The plasmids GFP-Trp53, flag-TAZ, and HA-Ub were from Addgene (USA).
3
Western Blot Analysis of Protein Expression
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Proteins were extracted, and their concentrations were measured with the BCA protein assay kit (Beyotime, Shanghai, China). The proteins were then electrophoresed by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Next, the membranes were blocked in 5% bovine serum albumin (BSA) and incubated overnight at 4 °C with specific primary antibodies (1:1000). Rabbit anti-PTPRB (Abcam, Cambridge, UK), GAPDH, N-cadherin, E-cadherin, vimentin, p-LATS1, p-YAP, p-TAZ, LATS1, YAP, and TAZ (Cell Signaling Technology) antibodies were used. After that, the membranes were incubated with the secondary antibody (1:5000) at room temperature for 2 h. Reacting bands were visualized using ECL reagent (Thermo Fisher Scientific), and the density of protein bands was semi-quantified using ImageJ.
4
Protein Extraction and Western Blotting Protocol
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Total protein of cells was extracted by using RIPA lysis buffer (Solarbio, Beijing, China) containing protease inhibitors and centrifuged at 12 000g for 20 min at 4°C. The protein concentration was determined by BCA protein assay (Beyotime, Shanghai, China). Subsequently, protein (50 μg) was separated by SDS-PAGE and then transferred onto PVDF membranes. Then, the membranes were blocked and incubated with primary antibodies (ISLR, MMP2, Vimentin, PDLIM4, E-cadherin, N-cadherin, Snai2 and GAPDH were purchased from Sigma-Aldrich; YAP, p-YAP, TAZ, p-TAZ and LaminB1 were purchased from Cell Signaling Technology, Danvers, Massachusetts, USA) overnight at 4°C and incubated with the second antibody for 1 h at 37°C. After washing with TBST, ECL enhanced chemiluminescence detection system (Thermo Scientific, Rockford, Illinois, USA) was used to detect the protein bands.
5
Proteomic Analysis of Lung Lysates
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Protein samples of lung lysates were separated on 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with the Bio-Rad system followed by transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). After blocking the PVDF membranes with 5% milk in TBST, primary antibodies of YAP (1:500), p-YAP (1:1000), T1α (1:1000) (Abcam, Cambridge, UK), TAZ (1:1000), p-TAZ (1:1000), sirtuin 1 (SIRT1) (1:1000), p53 (1:1000) (Cell Signaling Technology, Massachusetts, USA), SPC (1:5000) (SAB, Maryland, USA), and β-actin (1:5000) (GeneTex, California, USA) were used. The membranes were then incubated with anti-rabbit (1:5000) or anti-mouse (1:5000) secondary antibodies, followed by incubation with an enhanced chemiluminescence (ECL) reagent. Images were taken with the BioSpectrum Imaging System (UVP, Upland, CA, USA) and analyzed using Image-Pro vers. 4 (Media Cybernetics, Rockville, MD, USA).
6
Western Blot Analysis of EMT Markers
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Whole-cell lysates were extracted in a lysis buffer (Beyotime, Shanghai, China) and were separated by 10% SDS-PAGE, then transferred to a polyvinylidene uoride membranes (Millipore, Billerica, MA, USA) by using a wet transfer apparatus (Bio-Rad, German). Blots were blocked in 5% (w/v) skim milk for 2 h at room temperature and then incubated with antibodies against snail, Vimentin, MMP1 (Abcam, Cambridge, UK), P-TAZ, P-YAP, TAZ, YAP, AMPK, p-AMPK (Cell Signaling Technology, USA) and GAPDH (ZSGB-BIO, China) overnight at 4 °C. The HRP-coupled anti-rabbit secondary antibody (ZSGB-BIO, China) was used at a nal dilution of 1:1000, visualized with an enhanced chemiluminescence (ECL) detection system (Thermo Scienti c, Waltham, MA, USA).
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