After adding 200 μL of the lysate to the 6-well plate, it was allowed to stand for 20 minutes on ice, and the liquid was collected and centrifuged (13,000 rpm, 15 min); then, the supernatant was collected. The concentration of the protein was determined by the bicinchoninic acid (BCA) (Camilo Biological, Nanjing, China) method and quantified. The protein was separated using a 10% sodium dodecyl sulfate-polyacrylamide gel, then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) for 2 hours at 4°C. 5% skim milk was prepared with Tris-buffered saline with Tween-20 (TBST) to block the specific antigen for 2 hours. After washing with TBST for 1 minute, PVDF membranes were incubated with a specific primary antibody (SOD1, Abcam, Rabbit; 1 : 3000; SOD2, Abcam, Rabbit, 1 : 3000; GPX1, US 1 : 1000, CST; GPX3, US 1 : 1000, CST; Bcl-2, Abcam, Rabbit, 1 : 2000; Bax, Abcam, Rabbit, 1 : 500; Sirt1, Bioworld, mouse, 1 : 500, China; P53, Abcam, Rabbit, 1 : 2000; ROCK1, Abcam, Rabbit, 1 : 500; ROCK2, Abcam, Rabbit, 1 : 1000; MYPT-1, Abcam, Rabbit, 1 : 1000; GAPDH US 1 : 1000 CST) at 4°C overnight. The next day, TBST was washed for 30 minutes. Specific proteins were detected by secondary antibodies and observed by the electrochemiluminescence (ECL) (Pierce, Rockford, IL, USA) system.
Mypt 1
Manufactured by Abcam
Sourced in China
MYPT-1 is a regulatory subunit of the protein phosphatase 1 (PP1) enzyme complex. It acts as a targeting subunit that directs the PP1 catalytic subunit to specific substrates and cellular locations. MYPT-1 plays a role in the regulation of various cellular processes such as smooth muscle contraction, cell migration, and cytokinesis.
Automatically generated - may contain errors
Lab products found in correlation
Mypt1 p thr696, by Merck Group (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg and goat anti mouse igg secondary antibodies, by Thermo Fisher Scientific (2 mentions)
α tubulin, by Biosynth (2 mentions)
Hif 2α, by Novus Biologicals (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Rock2, by Abcam (1 mentions)
Rock1, by Abcam (1 mentions)
Sirt1, by Bioworld Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
P mypt1, by Santa Cruz Biotechnology (1 mentions)
Loading buffer, by Biosharp (1 mentions)
Phospho mlc, by Cell Signaling Technology (1 mentions)
Non fat dried milk, by Merck Group (1 mentions)
Bca assay kit, by Biosharp (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
Protein phosphatase inhibitor, by Solarbio (1 mentions)
6 protocols using mypt 1
1
Protein Expression and Western Blot Analysis
2
Western Blot Analysis of Cellular Signaling
3
ROCK1 Phosphorylation of MYPT1 Substrate
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ROCK1 phosphorylates the myosin phosphatase target subunit 1 (MYPT1)27 (link), known as the myosin-binding subunit (MBS) of myosin light-chain phosphatase, a Thr853. Myosin phosphatase target subunit 1 (p-MYPT1) reflects the Rho-kinase activity28 (link) as a substrate protein. In brief, cell lysates and kidney cortical tissue extracts were analyzed using western blot analysis and rabbit polyclonal antibodies against p-MYPT1 (Santa Cruz, Dallas, TX, USA) and MYPT1 (Abcam, Cambridge, MA, USA). The ratio of p-MYPT1 and MYPT1 exhibited ROCK1 activity.
4
Western Blot Analysis of Cellular Signaling
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After treatments, cells were lysed and Western blot was conducted as previously described55 (link). Proteins were visualized using primary antibodies recognizing HIF1α (BD Biosciences, #610959, San Jose, CA), HIF2α (Novus Biological, #NB100–122, Littleton, CO), α-tubulin (Fitzgerald, #10R-842, North Acton, MA), MYPT1-P Thr696 (EMD Millipore, #ABS45, Temecula, CA), MYPT1, (Abcam, #ab32393, Cambridge, MA), Cleaved Caspase 3, VHL (Cell Signaling, #9661, #2738, Danvers, MA), ROCK1, ROCK2 (Thermo Scientific, #PA5–22262, #PA5–21131, Grand Island, NY); and Horseradish peroxidase conjugated Goat anti-Rabbit IgG and Goat anti-Mouse IgG secondary antibodies (Thermo Scientific, #31460, #31430). Blots were imaged using a Bio Rad ChemiDoc XRS+ (BioRad, Hercules, CA).
5
Western Blotting for Protein Expression
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Western blotting was performed as previously described (29 (link)). Total protein was extracted in radioimmunoprecipitation assay (RIPA) buffer and phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology, Shanghai, China) at a ratio of 100:1. The protein concentration was measured by a BCA assay kit (Biosharp, AnHui, China), and the protein was loaded onto a 5 × sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel; a loading buffer (Biosharp, Anhui, China) at a volumetric ratio of 5:1 was used.
After denaturation and SDS–PAGE electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% non-fat dried milk (Sigma, Shanghai, China) and incubated with primary antibodies including anti-ROCK1, anti-RhoA, anti-ROCK2, anti-α-SMA, anti-eNOS, anti-iNOS anti-ET-1, anti-CD31 phospho-MYPT-1, MYPT-1 (Abcam, Shanghai, China), phospho-MLC, MLC (Cell Signaling Technology), and GAPDH (Proteintech, WuHan, China) overnight at −20°C. The PVDF membranes were washed with TBST and then with secondary antibodies. Finally, the membranes were washed again and incubated in chemiluminescent ECL substrate (Fisher). The images were analyzed by ImageJ software, and the results were normalized to those of GAPDH. Analysis was performed using ImageJ software.
After denaturation and SDS–PAGE electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% non-fat dried milk (Sigma, Shanghai, China) and incubated with primary antibodies including anti-ROCK1, anti-RhoA, anti-ROCK2, anti-α-SMA, anti-eNOS, anti-iNOS anti-ET-1, anti-CD31 phospho-MYPT-1, MYPT-1 (Abcam, Shanghai, China), phospho-MLC, MLC (Cell Signaling Technology), and GAPDH (Proteintech, WuHan, China) overnight at −20°C. The PVDF membranes were washed with TBST and then with secondary antibodies. Finally, the membranes were washed again and incubated in chemiluminescent ECL substrate (Fisher). The images were analyzed by ImageJ software, and the results were normalized to those of GAPDH. Analysis was performed using ImageJ software.
6
Lung Tissue Protein Extraction and Western Blot Analysis
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Total proteins were extracted from lung tissues using the RIPA buffer (Solarbio, Beijing, China) in the presence of Protease Inhibitor Cocktail (Solarbio) and Protein Phosphatase Inhibitor (Solarbio), loaded on an SDS-PAGE gel, and electroblotted onto PVDF membrane. The primary antibodies were applied for overnight in 5% BSA at 4°C. WB were analyzed using ImageLab (Bio-Rad, Berkeley, CA). Primary antibodies: β-actin antibody (Abcam, Cambridge, UK); NF-κB p65 (Abcam); p-NF-κB p65 (Abcam); phospho-myosin phosphatase target subunit 1 (p-MYPT1; Abcam); MYPT1(Abcam); RhoA (Abcam); peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α; Abcam); nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4; Abcam); intercellular adhesion molecule (ICAM-1; Abcam); transforming growth factor-beta1 (TGF-β1; Abcam); COL3A1 (Abcam); COL1A2 (Abcam).
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