Protein Extraction kit (catalog no. NBP2-37853; Novus Biologicals LLC, Littleton, CO, USA) was used to extract total protein from in vitro cultured cells. A bicinchoninic acid Protein Quantification kit (catalog no. ab102536; Abcam, Cambridge, UK) was used to determine protein concentrations with all steps performed in strict accordance with the manufacturer's protocol. Protein samples were denatured and subjected to SDS-PAGE (10% gels; 45 µg per lane). Following protein transfer onto polyvinylidene difluoride membranes, membranes were blocked in 5% fat-free milk in PBS at room temperature for 2 h. The primary antibodies used were rabbit anti-human ROCK1 (1:1,400; catalog no. ab97592; Abcam) and rabbit anti-human GAPDH, (1:1,400; catalog no. ab9485; Abcam) and the incubation was performed at 4°C for 15 h. A secondary incubation was performed at 24°C for 2 h using horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:1,200; catalog no. MBS435036; MyBioSource, Inc., San Diego, CA, USA). An ECL™ Western Blotting Analysis system (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to develop signals. ImageJ software (version 146; National Institutes of Health, Bethesda, MD, USA) was used to normalize all the data.
Ab97592
Manufactured by Abcam
Sourced in United States
Ab97592 is a lab equipment product offered by Abcam. It is a device designed for laboratory use, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (2 mentions)
Ecl western blotting analysis system, by Merck Group (1 mentions)
Protein extraction kit, by Novus Biologicals (1 mentions)
Ckx53, by Olympus (1 mentions)
Ab63856, by Abcam (1 mentions)
Ab192256, by Abcam (1 mentions)
Ab54835, by Abcam (1 mentions)
Ab52488, by Abcam (1 mentions)
Ab209847, by Abcam (1 mentions)
Ab125025, by Abcam (1 mentions)
Ab115730, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Ab37168, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Bio-Rad (1 mentions)
Mbs435036, by MyBioSource (1 mentions)
Bca assay, by Merck Group (1 mentions)
Ab22555, by Abcam (1 mentions)
Ripa solution, by Merck Group (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
8 protocols using ab97592
1
Western Blot Analysis of ROCK1 Protein
2
Immunohistochemistry Analysis of Valve Biomarkers
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Immunohistochemistry detection of Rho A, ROCK1, ROCK2, GLUT1, HK2, PDK1, PFK1, LDHA, OPN and RUNX2 in human aortic valve leaflets was performed. 4% PFA-fixed paraffin sections (5μm thick) were deparaffinized using dewaxed solution and hydrated using decreasing concentration of ethyl alcohol (100%, 90%, 80%, 70%, 60%), and then incubated at 65 °C for 20 min in antigen unmasking solution using PT Module-Lab Vision (Thermo Fisher Scientific) for antigenic retrieval. Following several washes (3 x 5 min) in PBS, paraffin sections were blocked for 30 min using 5% bovine serum albumin (BSA). After blocking, primary antibodies against Rho A (abcam, ab54835), ROCK1 (abcam, ab97592), ROCK2 (abcam, ab125025), GLUT1 (abcam, ab115730), HK2(abcam, ab209847), PDK1 (abcam, ab202468), PFK1 (CST, #8164), LDHA (abcam, ab52488), OPN (abcam, ab63856) and RUNX2 (abcam, ab192256) were diluted at optimized concentrations in 1% BSA and incubated with the sections in a wet box overnight at 4 °C. Subsequently, appropriate secondary antibodies were applied for 30 min at room temperature. Following secondary antibody incubation, the positive staining was detected using an UltraSensitive SP IHC Kit and DAB Kit, and hematoxylin staining was used for nuclear counterstaining. Images were visualized using a microscope (CKX53, Olympus), and Image J software was applied for data quantification.
3
Quantifying ROCK1 Expression in lncRNA CCHE1 Knockdown
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Knockdown rate of lncRNA CCHE1 were reached before this experiment. RIPA (Bio-Rad) was used to isolate proteins. Proteins were separated using 12% SDS-PAGE gel. Western blotting was performed using conventional method. Primary antibodies included rabbit anti-human ROCK1 (1: 1200, ab97592, Abcam) and rabbit anti-human GAPDH antibody (1: 1200, ab37168, Abcam). IgG-HRP (1:1000, MBS435036, MyBioSource) was used as the secondary antibody. ECL (Sigma-Aldrich, USA) was used for signal development and data normalization was performed using Image J software.
4
Detection of ROCK1 Expression in SHP-77 Cells
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SHP-77 cells were harvested at 48 h of post-transfection, and ROCK1 expression was detected by Western blot. In brief, total proteins from 105 SHP-77 cells were extracted using 1 ml RIPA solution (Sigma-Aldrich) and quantified using BCA assay (Sigma-Aldrich). The same amounts of proteins were separated on 12% SDS-PAGE gels after denatured in boiling water for 15 min and transferred onto PVDF membranes. After blocked with 5% fat-free milk at 24 °C for 1 h, the membranes were incubated first with primary antibodies against GAPDH (1:1600, ab22555, Abcam) and ROCK1 (1:1600, ab97592, Abcam) at 4 °C for 12 h, then with HRP-labeled goat secondary antibody (IgG) (1:2000; ab6721; Abcam) at 24 °C for 1 h, and last with ECL Western Blotting Substrate (ab65623, Abcam) to develop signals. The signals were processed using Image J v1.46 software.
5
Western Blot Analysis of Hepatic Proteins
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Liver tissues or cultured cells were lysed in the commercial RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China) containing protease inhibitor cocktail followed by spinning down at 15,000 rpm for 12–15 min. The supernatant was saved to quantify the protein concentration using the commercial kit (Pierce BCA protein Assay kit, Thermo Fisher Scientific, MA, USA). Equal amounts of protein from each sample were loaded into the SDS-PAGE gels and separated through electrophoresis. The proteins in the gels were then transferred into the nitrocellulose membranes (Bio-Rad, CA, USA). The membranes were blocked in 3% BSA for 0.5 h at room temperature and then primary antibodies were added for overnight incubation at 4 °C. The next day, the membranes were washed with TBST and secondary antibodies were added for incubation for an additional 1 h at room temperature. Signals were detected using the commercial ECL kit (Sigma-Aldrich, MO, USA). The following primary antibodies were used: Anti-ROCK1 (1: 1000; ab97592, Abcam, USA); Anti-p-AMPK (1:500; 2535 S, Cell Signaling, USA). Anti-AMPK (1:1000; 2532, Cell Signaling, USA); Anti-SREBP1 (1:1000; ab28481, Abcam, USA); Anti-FAS (1:1000; 3180, Cell Signaling, USA); Anti-SCD1 (1:1000; ab236868, Abcam, USA); Anti-ACC1 (1:500; ab72046, Abcam, USA); Anti-CD36 (1:1000; ab252923, Abcam, USA); Anti-GAPDH (1:2000; ab9485, Abcam, USA).
6
Quantitative Protein Analysis via RIPA-ROCK1
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Radioimmunoprecipitation assay (RIPA) containing protease inhibitor was applied for extraction of total protein. 50 μg proteins of each sample were electrophoresed on 12% TruPAGE™ Precast Gels using Sigma-Aldrich® Dual Run and Blot System (Sigma) and transferred to Immobilon-E Polyvinylidene Fluoride Membrane (Sigma) through the Trans-Blot Turbo Transfer System (Bio-Rad). The non-specific proteins were blocked and the membranes were incubated with primary antibodies of ROCK1 (Abcam, Cambridge, UK; ab97592, 1:1000) or GAPDH (Abcam, ab128915, 1:1000) at 4 °C overnight. After the incubation of Goat Anti-Rabbit IgG H&L secondary antibody (ab205718, 1:3000), immunoreactive blots were visualized via Enhanced Chemiluminescence (ECL) Substrate (Bio-Rad). GAPDH were used as the internal reference, and the protein intensity was analyzed by ImageJ software (NIH).
7
Western Blot Analysis of Apoptosis Regulators
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MGC-803 and AGS cells were washed with PBS and lysed in RIPA lysis. After the centrifugation and quantification, protein lysates (30 μg) were loaded onto SDS-PAGE and then suffered from the membranes transfer using PVDF membranes purchased from Millipore (Billerica, MA, USA). The membranes were blocked with 5% non-fat milk, incubated with primary antibodies against Bax (ab199677, 1:1000 dilution, Abcam, Cambridge, MA, USA), Bcl-2 (ab196495, 1:5000 dilution, Abcam), ROCK1 (ab97592, 1:2000 dilution, Abcam) at 4°C, and then interacted with HRP- conjugated IgG (ab205718, 1:10,000 dilution, Abcam) for 2 h. The antibody against β-actin (ab227387, 1:10,000 dilution, Abcam) was used as loading control. The protein band signaling was developed by enhanced chemiluminescence solution (Beyotime) and grayscale value was analyzed by Image Lab software (Bio-Rad, Hercules, CA, USA).
8
Western Blot Analysis of ROCK1 and GAPDH
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RIPA solution (Invitrogen) and BCA assay (Invitrogen) were used to isolate and quantify protein samples. Denatured protein samples were separated by SDS-PAGE gels (10%) and gel transfer was performed using PVDF membranes. Membranes were blocked in FBS containing 5% non-fat milk. After that, primary antibodies of GAPDH (ab9483, Abcam) and ROCK1 (ab97592, Abcam) were incubate with membranes at 4°C for 18h. After that, at room temperature, membranes were further incubated with HRP (IgG) (ab6721, Abcam) for 2h. Signals were produced using ECL (Invitrogen) and Quantity One software was used for data quanti cation.
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