Ishikawa cells which had received the different treatments were scraped from the six-well plates and lysed with RIPA lysing buffer (Beyotime; Nantong, China) containing 1 mM PMSF and a protease inhibitor cocktail (Beyotime). The lysed cells from each treatment group were centrifuged at 16,000g for 20 min, and the supernatant fractions were collected. Next, a 20 μg sample of supernatant protein was separated by 8 % SDS-PAGE, and then semi-dry blotted onto PVDF (polyvinylidene fluoride) membranes (Millipore, Bedford, MA, USA). After being blocked for 2 h with TBST containing 5 % non-fat dry milk, the membranes were incubated overnight at 4 °C with a primary rabbit monoclonal antibody to integrin β1 (1:5000) or integrin β3 (1:5000); after which, they were incubated with the HRP-labeled secondary antibody (1:10,000; Cwbiotech, Beijing, China) for 1 h at room temperature. HRP-labeled β-actin (1:10,000; Sigma-Aldrich) was used as an internal control protein. The protein blots were developed using Beyo ECL Plus reagent (ThermoFisher Scientific; Waltham, MA, USA), and the results were recorded with a gel imaging system. The relative expression levels of integrins β1 and β3 in the different samples were calculated using a Gel-Pro-Analyzer (Media Cybernetics; Rockville, MD, USA).
Beyo ecl plus reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Beyo ECL Plus reagent is a chemiluminescent detection solution used for the quantitative analysis of proteins in Western blotting applications. The reagent is designed to produce a stable luminescent signal, allowing for sensitive and accurate detection of target proteins.
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2 protocols using beyo ecl plus reagent
1
Integrin Expression Quantification in Ishikawa Cells
2
Protein Expression Analysis Protocol
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The total proteins from different samples were extracted using a Total Protein Extraction kit according to the manufacturer's instructions (cat. no. WLA019; Wanleibo Co., Ltd., Beijing, China). GAPDH was used as an internal reference protein. The protein concentration of each sample was determined using the bicinchoninic acid method. An aliquot of protein (40 μg) from each sample subjected to 10% SDS-PAGE performed at 80 V for 2.5 h. The separated proteins were transferred onto polyvinylidene difluoride membranes, which were then washed with TBS-Tween for 5 min, and subsequently incubated with a powdered skim milk solution for 1 h. Subsequently, the membranes were incubated with primary antibodies against OPG (1:1,000), RANKL (1:500), VEGF (1:500) or GAPDH (1:500) at 4°C overnight. Following incubation, the membranes were washed four more times with TBS-Tween, and then incubated with secondary IgG-horseradish peroxidase antibodies [1:5,000; goat anti-mouse IgG (BA1050) and goat anti-rabbit IgG (BA1054), Wuhan Boster Biological Technology, Ltd.] for 45 min at 37°C. Following incubation, the membranes were washed six times with TBS-Tween, and the blots were developed using Beyo ECL Plus reagent (Thermo Fisher Scientific, Inc.). The images were recorded using a gel imaging system.
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