Anti mypt1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-MYPT1 is a primary antibody targeting the Myosin Phosphatase Target Subunit 1 (MYPT1) protein. MYPT1 is a regulatory subunit of the myosin phosphatase enzyme, which plays a role in the regulation of smooth muscle contraction. This antibody can be used in various research applications to detect and study the MYPT1 protein.

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6 protocols using anti mypt1

EZH2 and MYPT1 Immunoprecipitation

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Cells were collected on ice and lysed in 0.2% NP‐40 co‐IP buffer (10 × 10^‐3m Tris‐Cl, pH 7.4, 150 × 10^‐3m NaCl, 0.2% NP‐40, and 10% glycerol) supplemented with protease inhibitors and protein phosphatase inhibitor cocktail, incubated on ice for 15 min, and cleared by centrifugation at 13 200 rpm at 4 °C for 15 min. Total protein lysate (2–5 mg) was precleared with normal rabbit IgG for 2 h at 4 °C, and 1 mL precleared lysate was subjected to immunoprecipitation with EZH2 antibody (Cell Signaling Technology#5246) or MYPT1 antibody (Cell Signaling Technology #8574) as well as protein A/G agarose beads overnight at 4 °C. The immunoprecipitates were washed six times with co‐IP buffer, and then the washing buffer was completely removed from the beads. The beads were boiled in SDS sample buffer, and then the proteins were separated by SDS‐PAGE and transferred to nitrocellulose membranes. The blots were developed with anti‐MYPT1 (1:1000, Santa Cruz, sc‐514261) or with anti‐EZH2 (Cell Signaling Technology #5246 & #3147) antibodies.

Zhang L., Wang L., Hu X., Hou M., Xiao Y., Xiang J., Xie J., Chen Z., Yang T., Nie Q., Fu J., Wang Y., Zheng S., Liu Y., Gan Y., Gao Q., Bai Y., Wang J., Qi R., Zou M., Ke Q., Zhu X., Gong L., Liu Y, & Li D.W. (2022). MYPT1/PP1‐Mediated EZH2 Dephosphorylation at S21 Promotes Epithelial–Mesenchymal Transition in Fibrosis through Control of Multiple Families of Genes. Advanced Science, 9(14), 2105539.

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Seawater-Based Inflammatory Response Study

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Calcitriol (HPLC purity≥99%) and dexamethasone (HPLC purity≥98%) were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Seawater (osmolality 1300 mmol/L, PH 8.2, SW 1.05, NaCl 6.518 g/L, MgSO₄ 3.305 g/L, MgCl₂ 2.447 g/L, CaCl₂ 1.141 g/L, KCl 0.725 g/L, NaHCO₃ 0.202 g/L, NaBr 0.083 g/L) was prepared according to the major composition of the East China Sea provided by Chinese Ocean Bureau. Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-1β and IL-6 were obtained from R&D Systems (Minneapolis, MN, USA). Anti-RhoA, anti-MYPT1, anti-pMYPT1, anti-CD31 and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-NF-κB p65, anti-pNF-κB p65 antibodies were purchased from Cell Signaling Technology (Cell Signaling, MA, USA).

Zhang M., Dong M., Liu W., Wang L., Luo Y., Li Z, & Jin F. (2014). 1α,25-Dihydroxyvitamin D3 Ameliorates Seawater Aspiration-Induced Acute Lung Injury via NF-κB and RhoA/Rho Kinase Pathways. PLoS ONE, 9(8), e104507.

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Western Blot Protein Quantification

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Western blot assays were performed as described earlier [19 ]. A buffer was used to lyse cells or tissues, followed by 20 min of freezing at −20 °C. Insoluble debris from the sample was removed by centrifuging it at 12000×g at 4 °C for 20 min. Next, the supernatant samples were collected, and protein levels were calculated by the Bradford method (Bio-Rad Protein Assay). The gray value of Western blot was quantified and normalized to the internal reference (GAPDH or Actin) with ImageJ software (NIH). We utilized the following primary antibodies: anti-α-smooth muscle actin (1:1000, ab7817); anti-TRPC3 (1:1000, Alomone, ACC-016); anti-collagen I (1:1000, Cell Signaling Technology, ^#84336); anti-MLC (1:1000, Cell Signaling Technology, ^#3675); anti-pMLC (1:1000, Cell Signaling Technology, ^#3672); anti-MYPT1 (1:1000, Santa Cruz Biotechnology, Sc-51426); anti-pMYPT1 (Santa Cruz Biotechnology, Sc-33360); anti-Fibronectin (1:1000, Abcam, ab268020); anti-phosphorylated Ser695 pyruvate dehydrogenase E1a subunit (PDHE1a) anti-pPDHE1a (1:1000, Merck-Millipore) and anti-GAPDH from Santa Cruz Biotechnology.

Xia W., Wang Q., Lin S., Wang Y., Zhang J., Wang H., Yang X., Hu Y., Liang H., Lu Y., Zhu Z, & Liu D. (2023). A high-salt diet promotes hypertrophic scarring through TRPC3-mediated mitochondrial Ca2+ homeostasis dysfunction. Heliyon, 9(8), e18629.

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Lipid-Induced Signaling Pathway Analysis

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Palmitate and oleate were obtained from Sigma-Aldrich. DMEM medium was obtained from Gibco. FBS was purchased from the Zhejiang Tianhang Biological Technology. anti-MLCK, anti-MLC, anti-MYPT1, anti-p-MYPT1, anti-ERK, anti-p-ERK and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology. Anti-pMLC was obtained from Cell Signaling Technology. Anti-NOXA2/p67phox and anti-Rac family small GTPase 1 were purchased from Abcam.

Wang Y., Wang M., Yu P., Zuo L., Zhou Q., Zhou X, & Zhu H. (2020). MicroRNA-126 Modulates Palmitate-Induced Migration in HUVECs by Downregulating Myosin Light Chain Kinase via the ERK/MAPK Pathway. Frontiers in Bioengineering and Biotechnology, 8, 913.

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Western Blot Analysis of Mitochondrial Proteins

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Western blotting was conducted on protein extractions that were obtained from the cell lysates. The protein concentrations were measured using a bicinchoninic acid (BCA) kit. Lysate proteins were separated using 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred electrophoretically to a nitrocellulose membrane. The membranes were blocked for 1 hour at room temperature using 5% nonfat milk in tris-buffered saline (TBS), then incubated at 4°C overnight with the following primary antibodies: anti-MYPT1 (1:1000; this and all subsequent antibodies were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Drp1 (1:1000), anti-Mfn1 (1:1000), anti-PGC-1α (1:1000), anti-NRF-1 (1:1000), anti-pro-caspase 3 (1:1000), anti-pro-caspase 9 (1:1000), and beta-actin (1:1000). After washing in TBS-Tween20, the membranes were incubated with secondary antibodies for 2 hours at room temperature. After washing, the membranes were incubated with enhanced chemiluminescence (ECL) reagents and scanned using a Bio-Rad Electrophoresis Image Analyzer (Bio-Rad, Hemel Hampstead, UK).^{19 (link),20}

Su Y., Hu L., Wang Y., Ying G., Ma C, & Wei J. (2021). The Rho kinase signaling pathway participates in tubular mitochondrial oxidative injury and apoptosis in uric acid nephropathy. The Journal of International Medical Research, 49(6), 03000605211021752.

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ROCK Inactivation of Myosin Phosphatase

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The total protein was measured via a bicinchoninic acid assay kit (Pierce) after sonication. 15 ug per well protein was loaded on 7.5% polyacrylamide gels, transferred onto nitrocellulose paper, and stained with primary and secondary antibodies. Reactive bands were visualized using the SuperSignal ECL Western blotting detection kit (Pierce), and the densitometry was obtained via the ImageJ software. In this study, myosin phosphatase was inactivated by ROCK through the specific phosphorylation of myosin phosphatase target subunit 1 (MBS or MYPT1) at Thr-853. This resulted in an increase in the phosphorylated content of the 20-kDa myosin light chain. Therefore, the ratio of pMBS to MBS was used to represent the ROCK activity. The primary monoclonal antibodies used in the western blot were anti-MYPT-1, phosphor T853-MYPT1 and IL-37, which were all from Santa Cruz Biotechnology.

Yang T., Fang F., Chen Y., Ma J., Xiao Z., Zou S., Zheng N., Yan D., Liao S., Chen S., Fang H., Yu C., Liu J, & Dong M. (2016). Elevated plasma interleukin-37 playing an important role in acute coronary syndrome through suppression of ROCK activation. Oncotarget, 8(6), 9686-9695.

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