Protein extracts of mouse ventricles or fibroblasts were prepared in RIPA buffer containing protease-inhibitor and phosphatase-inhibitor tablets (Roche, Vienna, Austria). After mechanical disruption of tissue samples, equivalent amounts of protein were separated on a SDS polyacrylamide gel, followed by electro-transfer to nitrocellulose membrane. Nonspecific antibody binding was blocked by incubation in 5% (m/v) non-fat dry milk powder in TBS-T (20 mM Tris-Cl, pH 7.5, 150 mMNaCl, 0.1% (v/v) Tween 20) at room temperature for 1 h. Then, samples were incubated overnight at 4°C with antibodies: anti-ABCG2 (#ab3380, Abcam) and anti-α-tubulin (#ab4074, Abcam) for ventricular tissues, and anti-ABCG2 (#AV43649, Sigma-Aldrich, Saint Louis, USA) and anti-GAPDH (#sc-25578, Santa Cruz Biotechnology, Heidelberg, Germany) for fibroblasts. After 1 h incubation in room temperature with peroxidase-labeled secondary antibody (Pierce Biotechnology, Rockford, USA), specific immunoreactive signals were detected using Enhanced ChemiLuminescence kit (Amersham Biosciences, Buckinghamshire, UK) or West Pico Substrate (Thermo Scientific, Rockford, USA).
Protease inhibitor and phosphatase inhibitor tablets
Manufactured by Roche
Sourced in United States
Protease inhibitor and phosphatase inhibitor tablets are laboratory reagents used in biochemical and molecular biology research. They inhibit the activity of proteases and phosphatases, which are enzymes that cleave peptide bonds or remove phosphate groups, respectively. These inhibitors help preserve the integrity of proteins and other biomolecules during experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ab4074, by Abcam (1 mentions)
Ab3380, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
West pico substrate, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti abcg2, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Ab98887, by Abcam (1 mentions)
Irdye 800cw donkey anti rabbit secondary antibody, by LI COR (1 mentions)
Image studio version 5, by LI COR (1 mentions)
Odyssey infrared scanner, by LI COR (1 mentions)
Lopac1280, by Merck Group (1 mentions)
Phospho histone h2a x, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Donkey anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Donkey anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
β catenin ctd, by BD (1 mentions)
β catenin ntd, by Abcam (1 mentions)
Image studio lite software, by LI COR (1 mentions)
6 protocols using protease inhibitor and phosphatase inhibitor tablets
1
Immunoblotting Analysis of ABCG2 Transporter
2
Protein Profiling of HRG-β1 Stimulated Cells
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Cell lines were seeded at 7,500 cells per well in 384-well culture plates in RPMI containing 4% FBS. 48-hour post plating, cells were stimulated (or not) with 2 nM HRG-β1 for four hours. At harvest, cells were placed on ice, and 70 μl RIPA lysis buffer (Sigma-Aldrich) supplemented protease inhibitor and phosphatase inhibitor tablets (Roche) was added to each well. The plates were stored at -80°C until analysis. On the first day of protein profiling, the lysates were thawed at 4°C and centrifuged at 4000 rpm for 10 minutes. The supernatant was used for further analysis with multiplex Luminex protein assays as described below.
3
Western Blot Analysis of Retinal Rhodopsin
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Eyes were dissected in cold PBS and retinas were lysed in lysis buffer (100 µL per retina) composed of 20 mM Tris-HCl, pH 8, 150 mM NaCl, 2.5 mM EDTA, 10% glycerol, 0.5% Triton X-100, 0.01% Nonidet P-40 substitute, protease inhibitor and phosphatase inhibitor tablets (Roche Diagnostics, Indianapolis, IN, USA). Blots were probed with antibodies against rhodopsin (ab98887; Abcam, Cambridge, MA, USA) and β-actin (4970L; Cell Signaling Technology, Danvers, MA, USA). Immunoblots were visualized using IRDye 800CW donkey anti-rabbit secondary antibody (925-32213; Li-Cor Biosciences, Lincoln, NE, USA) and IRDye 680CW donkey anti-mouse secondary antibody. Membranes were scanned with an Odyssey infrared scanner (Li-Cor Biosciences). Densitometric analysis was performed using image studio version 5.2 (Li-Cor Biosciences). For the quantitation, 4 independent proteins blots were used and each lane represents retinal lysates from different animals. Six to eight animals were used per genotype for the normoxia and hyperoxia conditions.
4
LOPAC®1280 Compound Screening in Neuronal Cultures
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At DIV14, neuronal cultures were treated with specific compound of interest from the Library of Pharmacologically Active Compounds (LOPAC®1280) (Sigma-Aldrich, St. Louis, MO, USA) assortment. Allotments of all 1,280 compounds were provided by MD Anderson Cancer Center (Houston, TX, USA). Compounds were diluted to 10 uM in fresh Neurobasal media and dilutions were added directly to cultures for 6 hours of incubation. Neurons were lysed with Tris Lysis Buffer from MSD supplemented with Protease inhibitor and Phosphatase inhibitor tablets from Roche.
5
Colorectal Adenocarcinoma Protein Analysis
6
Western Blot Analysis of Cell Junctions
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Cells were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor tablets (Roche, IN, USA). Cell lysates were cleared by centrifugation and protein concentration determined by Bio-Rad Protein Assay kit (Bio-Rad Laboratories, CA, USA). 5–30 µg (as indicated in the figure legends) of total protein in SDS sample buffer was loaded per lane and separated on NuPAGE Tris-Acetate precast polyacrylamide gels (Life Technologies). Detection of proteins was done with the following primary Abs and dilutions: β-catenin CTD (BD Transduction Laboratories, #610153, 1∶2000), γ-catenin (BD Transduction Laboratories, #610253, 1∶5000) β-catenin NTD (Abcam, ab32572, 1∶5000), E-cadherin (BD Transduction Laboratories, #610181, 1∶4000), α-Catenin (BD Transduction Laboratories, #610193, 1∶500), p120-catenin (BD Transduction Laboratories, #610133, 1∶2000), Actin (Sigma-Aldrich, A2066, 1∶2000). Secondary Abs were: donkey anti-mouse IgG-HRP (Santa Cruz Biotechnology, CA, USA, sc-2314, 1∶5000), donkey anti-rabbit IgG-HRP (Santa Cruz biotechnology, sc-2313, 1∶5000). Signals were developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) and quantification of signal intensities was done using the Image Studio Lite software (LI-COR, NB, USA).
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