Anti mypt1

Manufactured by Abcam

Sourced in United States

Anti-MYPT1 is a lab equipment product that functions as an antibody. It is used for the detection and analysis of the MYPT1 protein in various experimental and research applications.

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Lab products found in correlation

Anti rhoa, by Cell Signaling Technology (1 mentions) Anti mlc, by Cell Signaling Technology (1 mentions) Phospho mlc, by Cell Signaling Technology (1 mentions) Anti ampk, by Abcam (1 mentions) Phospho mypt1, by Cell Signaling Technology (1 mentions) Phospho ampk, by Cell Signaling Technology (1 mentions) Anti o glcnac, by Thermo Fisher Scientific (1 mentions) Mounting media, by Agilent Technologies (1 mentions) Anti tubulin dm1a, by Merck Group (1 mentions) Anti myosin iia, by Merck Group (1 mentions) Alexa fluor 488 or 568 phalloidin, by Thermo Fisher Scientific (1 mentions) Anti gm130, by Cell Signaling Technology (1 mentions) Anti paxillin, by BD (1 mentions) Ix73 inverted fluorescence microscope, by Olympus (1 mentions)

3 protocols using anti mypt1

Aortic Protein Expression Analysis

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Proteins (60 µg) extracted from aortas or VSMCs were separated by electrophoresis on
10% polyacrylamide gels and transferred to nitrocellulose membranes. Nonspecific
binding sites were blocked with 5% skim milk in Tris-buffered saline solution with
10% Tween for 1 h at 24°C. Membranes were then incubated with antibodies overnight at
4°C. Anti-O-GlcNAc (CTD 110.6, 1:2000; Pierce Biotechnology, USA), anti-AMPK (#80039,
1:1000; Abcam, USA), anti-protein kinase CPI-17 (#32213, 1:1000; Abcam, USA),
anti-MYPT-1 (#2634), anti-rho-kinase (ROCK)-α (#8236), anti-ROCK-β (#4035), anti-MLC
(#8505) and anti-RhoA (#2117) (all 1:1000; Cell Signaling, USA, or BD Biosciences
Transduction Laboratories, USA) were used. Immunoblots for nonphosphoproteins were
carried out on the same membranes used to evaluate the phosphorylated (phospho-)
forms: phospho-MYPT-1 (Thr⁸⁵³), phospho-CPI-17 (Thr³⁸),
phospho-MLC (Thr¹⁸/Ser¹⁹), and phospho-AMPK
(Thr¹⁷²), (1:500; Cell Signaling, USA). After incubation with secondary
antibodies, signals were developed for chemiluminescence, visualized by
autoradiography, and quantified densitometrically. Results were normalized to
beta-actin protein (#A5316, 1:10000; Sigma-Aldrich, Inc., USA), or to the total form
of each phosphorylated protein, and reported as arbitrary units.

Lima V.V., Lobato N.S., Filgueira F.P., Webb R.C., Tostes R.C, & Giachini F.R. (2014). Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain. Brazilian Journal of Medical and Biological Research, 47(10), 826-833.

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Immunofluorescence Analysis of Cytoskeletal Proteins

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U2OS cells plated on fibronectin-coated (15μg/ml) coverslip were either pre-extracted in 0.25% paraformaldehyde, PFA (16% stock solution Electron Microscopy Science) and 0.05% triton in cytoskeleton buffer (CB; 10 mM MES 6.1, 138 mM KCl, 3mM MgCl, 2 mM EGTA) for 1 min at 37°C, and fixed in 4% PFA in CB for 20 min, or fixed in 4% PFA in CB for 20 min at 37°C. After fixation, coverslips were permeabilized in 0.5% Triton X-100 in CB for 5 min, free aldehydes were reacted with 100 mM glycine, washed in TBS, and blocked in blocking solution (2% BSA TBS-T) for 1 hr. Cells were incubated with primary antibodies (anti-MARK2 (1:250, Abcam), anti-tubulin DM1A (1:500, Sigma), anti-GM130 (1:500 Cell Signaling), anti-S19-PMRLC (1:200, Cell Signaling), anti-myosin IIA (1:400; Sigma Aldrich,), anti-paxillin (1:500; BD Biosciences, San Jose, CA) or anti-MYPT1 (1:250, Abcam) diluted in blocking solution, washed 3 times 10 min each in TBS-T and then incubated with fluorophore-conjugated secondary antibodies (1:400; Jackson ImmunoResearch Laboratories) and Alexa Fluor 488 or 568 phalloidin (1:400; Invitrogen), washed again and mounted on slides with mounting media (Dako, Pathology Products, Carpinteria, CA), or in TBS supplemented with n-propylgalate for TIRF imaging.

Pasapera A.M., Heissler S.M., Eto M., Nishimura Y., Fischer R.S., Thiam H.R, & Waterman C.M. (2022). MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization.. Current biology : CB, 32(12), 2704-2718.e6.

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Immunohistochemical Scoring of Cytoplasmic Proteins

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The slices were first baked in an incubator at 70°C for 30 min before dewaxing. and rehydrating them. Then the slides were soaked twice for 5 min in xylene, followed by washing twice with 100% ethanol (2 min each time), and washing twice with 95% ethanol (2 min each time). Trypsin was used to retrieve antigens and SP staining was performed. The primary antibodies including anti-CPI17 α (1:100; Abcam), anti-MYPT1 (1:100; Abcam) and anti-MYL12B (1:200; Abcam) antibodies were added and the samples were incubated overnight at 4°C. After washing the next day, secondary antibody (p6261; Abcam) was added and color development was performed (DAB chromogen for 1 ml substrate). An Olympus inverted fluorescence microscope (IX73) was used to capture images.
Three independent pathologists observed the slices, selecting five representative visual fields (10×40 times) from each slide. A scoring system gave 1 point to samples that constituted the number of cytoplasmic-positive cells accounting for <25% of the total number of cells (weak positive ±), 2 points when the percentage of positive cells was between 25 and 50% (+), 3 points for a 50–75% positivity rate (++), and 4 points for positivities >75% (+++). Additionally, the intensity of the color was represented by (±), (+) and (++) corresponding to 1, 2 and 3 points. The scores from the two methods were multiplied to get the final score.

Yin Y., Li Y., Pan J., Tang R., Zhu J., Qin Z., Xu X, & Wang J. (2017). Expression of MYPT1, CPI-17 and MLC20 in ileum of neonatal mouse NEC model and its significance. Experimental and Therapeutic Medicine, 14(3), 2221-2227.

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