As previously described [9 (link), 23 (link), 24 (link), 25 (link), 26 (link), 27 (link)], Western blots were used to determine protein expression in human mesenteric arteries. Briefly, mesenteric artery samples were homogenized in SDS sample buffer, and total extracted protein was then resolved by SDS-PAGE using a Bis-Tris buffer and 7.5% gels. For the Western blots, we used the actin band on the Coomassie stained gels to normalize protein loading. After SDS-PAGE, proteins were transferred onto a Hybond™ (GE Healthcare) membrane. MYPT1, LZ + MYPT1, and actin were visualized using appropriate antibodies; a rabbit polyclonal anti-MYPT1 (Cell Signaling Cat#: 2634), a monoclonal mouse anti-LZ + MYPT1 [23 (link)], and a rabbit polyclonal anti-actin (A2066, Sigma). Following washing, the blots were incubated with appropriate antibodies, scanned on a Kodak imager, and analyzed using ImageQuant TL software. The density of MYPT1 was divided by the actin signal to control for relative expression, and then indexed to the level in controls, set as 1. To normalize the data between blots, one sample was chosen as a standard, which was loaded on every gel [28 (link)].
Anti mypt1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-MYPT1 is a primary antibody product used for detection and analysis of MYPT1 (Myosin Phosphatase Target Subunit 1) in biological samples. MYPT1 is a regulatory subunit of the protein phosphatase 1 (PP1) holoenzyme, which plays a key role in the regulation of myosin light chain phosphorylation and cellular processes such as smooth muscle contraction. The Anti-MYPT1 antibody can be used in various applications, including western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of MYPT1 in different cell types and tissues.
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Lab products found in correlation
Anti p mypt1, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti phospho mypt1, by Cell Signaling Technology (2 mentions)
Proteinase and phosphatase inhibitor cocktail, by Roche (1 mentions)
P mypt1, by Abcam (1 mentions)
Anti rhoa, by Cell Signaling Technology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by Abcam (1 mentions)
Protein loading buffer, by Transgene (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Anti lats1, by Cell Signaling Technology (1 mentions)
Anti p yap, by Cell Signaling Technology (1 mentions)
Anti p lats1, by Cell Signaling Technology (1 mentions)
Anti histone h3, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Anti pcreb, by Cell Signaling Technology (1 mentions)
Anti yap, by Cell Signaling Technology (1 mentions)
Anti creb, by Cell Signaling Technology (1 mentions)
9 protocols using anti mypt1
1
Quantifying Arterial Protein Expression Using Western Blot
2
Western Blot Analysis of Lung and MLVEC Proteins
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For western blotting, the lung tissues and MLVECs were homogenized in cold RIPA lysis buffer (Beyotime Institute of Biotechnology) containing proteinase and phosphatase inhibitor cocktail (Roche Diagnostics). Equal amounts of protein extract (30-40 µg) were separated by 8-12% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). After blocking in 5% non-fat dry milk for 2 h at room temperature, the membranes were probed overnight at 4°C with anti-SPHK1 (cat. no. ab71700; 1:500; Abcam), anti-phosphorylated (p)-myosin phosphatase target subunit 1 (p-MYPT1; cat. no. 5163S; 1:1,000; Cell Signaling Technology, Inc.), anti-MYPT1 (cat. no. 2634S; 1:1,000; Cell Signaling Technology, Inc.), anti-RhoA (cat. no. ab187027; 1:3,000; Abcam) and anti-β-actin (cat. no. sc-47778; 1:500; Santa Cruz Biotechnology, Inc.) antibodies. After washing, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab6721 and ab6728; 1:5,000; Abcam). The antibody-reactive bands were visualized via an enhanced chemiluminescence western blotting detection system (EMD Millipore). The membranes were visualized using the UVP Bio-Imaging system. Densitometric analysis was performed using Quantity One software (version 4.6; Bio-Rad Laboratories, Inc.).
3
Western Blot Analysis of Protein Expression
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Protein samples were diluted 1:5 with protein loading buffer (6 × Transgen Biotech, Beijing, China) and heated at 95 °C for 5 min. Protein extracts were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes, and blocked with 5% nonfat dry milk in TBST. The membranes were incubated with primary antibodies at 4 °C overnight and with the horseradish peroxidase (HRP)-conjugated secondary antibodies at 37 °C for 1 hour. Protein bands were visualized by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA) and imaged by a FluorChem M gel documentation system (ProteinSimple, San Jose, CA). The primary antibodies (anti-YAP, anti-p-YAP, anti-LATS1, anti-p-LATS1, anti-MYPT1, anti-p-MYPT1, anti-CREB, anti-p-CREB, anti-GAPDH, anti-α-tubulin, anti-histone H3) and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). CTGF antibody was obtained from Sigma-Aldrich (St Louis, MO, USA).
4
Western Blot Analysis of Signaling Proteins
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After 24 h drug treatment, total protein was extracted using lysis buffer contained 4% SDS, protease inhibitor cocktail and phosphatase inhibitor (Roche, US). Equal amount of total proteins was resolved using denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blot. Antibodies used in WB analyses include anti-p-MYPT1 (Cell Signaling, Cat. No.4563), anti-p-MLC (Cell Signaling, Cat.No.3671), anti-MLC (Cell Signaling, Cat.No.3672), anti-MYPT1 (Cell Signaling, Cat. No.2634), anti-p-Raf (Abcam, Cat. No. ab135559), anti-Raf (Abcam, Cat. No. ab137435), anti-p-ERK (Santa Cruz, Cat. No. sc-16,982), anti-ERK (Santa Cruz, Cat. No. sc-292,838), anti-Ras(Q61L) antibody (NewEast Biosciences, Cat. No. NEBA10195) and anti-β-actin (Santa Cruz, Cat. No. sc-130,656). Immunoblots shown in the accompanying figures are representative of three independent experiments.
5
Molecular Markers of Tight Junction Regulation
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Primary antibodies for Western blot include anti–NKA β1 subunit (Upstate, 05-382), anti-occludin (Invitrogen, 71-1500), anti–zo-1 (Invitrogen, 61-7300), anti–zo-2 (Invitrogen, 71-1400), anti-actin (MilliporeSigma, A2066), anti-GAPDH (MilliporeSigma, CB1-001), anti–NKA β2 subunit (Abcam, ab185210), anti-DDK (OriGene, TA50011-100), anti-MRCKα (Santa Cruz Biotechnology Inc., sc-374568), anti-MYPT1 (Cell Signaling Technology, 2634S), anti–phospho-MYPT1 (Thr696, Cell Signaling Technology, 5163S), anti-MLC2 (Cell Signaling Technology, 3672S), and anti–phospho-MLC2 (Ser19, Cell Signaling Technology, 3671S). The primary antibodies for immunofluorescence include anti–occludin Alexa Fluor594 (Invitrogen, 331594), anti–zo-1-Alexa Fluor594 (Invitrogen, 339194), and anti-MRCKα (Thermo Fisher Scientific, PA1-10038). The inhibitor for MRCKα BDP5290 was purchased from Aobious. Myosin inhibitor blebbistatin was purchased from Abcam.
6
Multispecific Antibody Detection
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The following antibodies were used: anti-pSer19-MLC2 (3675), anti-Ki67 (12202), anti-cleaved caspase 3 (9661), anti-CD31 (77699), anti-pT696-MYPT1 (5163), anti-MYPT1 (2634), anti-pY461-Src (6943), anti-pT202/Y204ERK1/2 (4370), anti-ERK1/2 (4695), anti-pT308-Akt1 (2965), anti-pS473-Akt1 (4060), anti-Akt (2920), anti-PP2A-C (2038), anti-PP2A-A (2041), anti-Shc (2432), anti-PI3 kinase p85 (4257), and anti-RhoC (3430) (Cell Signaling Technology); anti-β-actin (A5316) (Sigma-Aldrich); anti-PyMT (sc-53,481), anti-GST (sc-138), anti-Src (sc-8056), and normal rat IgG (sc-2026) (SantaCruz Biotechnology); and anti-RhoA (ARH03) (Cytoskeleton, Inc.).
7
Immunoblot Analysis of Hepatocytes
8
Western Blot Analysis of ROCK and NF-kB Signaling
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U937 cells were washed with PBS and lysed as described previously (27 (link)). Total protein concentration was quantified with Bradford Assay. Extracted proteins were separated by SDS-PAGE (100 V), and transferred to a nitrocellulose membrane (110 V, 90 min). Membranes were blocked in 5% (w/v) non-fat milk powder in TBS-T for 1 h at room temperature (RT), washed with TBS-T, and incubated with primary antibody overnight at 4°C. Membranes were washed and incubated with HRP-conjugated secondary antibodies for 2 h at RT, then washed and incubated with enhanced chemiluminescence (ECL) reagents (Sigma-Aldrich, USA) according to the manufacturer’s instructions. The primary antibodies used were anti-ROCK1 (Cell Signaling, USA #4035), anti-ROCK2 (Cell Signaling, USA #9029), anti-MYPT1 (Cell Signaling, USA #2634), anti-pMYPT1 (T696) (Cell Signaling, USA #5163), anti-p65 (Cell Signaling, USA #8242), anti-p-p65(Ser536) (Cell Signaling, USA #3033), anti-GAPDH (Cell Signaling, USA #2118), and anti-β-actin (Cell Signaling, USA #8457). Secondary antibodies used were anti-mouse-HRP (Santa Cruz Biotechnology, USA SC-516102) and anti-rabbit-HRP (Santa Cruz Biotechnology, USA SC-2357).
9
Rho Kinase Pathway Analysis
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Cells (HCASMCs or PBMC) were lysed in NETF buffer (NaCl 100 mmol/L, EGTA 2 mmol/L, Tris HCL 50 mmol/L, NaF 50 mmol/L, 1% NP-40, protease inhibitor and Ser/Thr phosphatase inhibitors [Sigma, cocktail 1]). Cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose membranes then incubated with specific antibodies. Immunoprecipitation of phosphotyrosine protein was performed using the 4G10 antibody (Upstate Biotechnology; Millipore, Molsheim, France) Assessment of the phosphorylation level of the Rho kinase target MYPT subunit 1 (P-MYPT) with a rabbit polyclonal antibody to phospho-MYPT (P-MYPT1; Santa Cruz Biotechnology, CliniSciences, Naterre, France). Antibody to Arhgef1 was purchased from Santa Cruz Biotechnology. Anti-MYPT-1 was purchased from Cell Signaling. ATR1 antibody was from Santa Cruz Biotechnology. Immunoreactive proteins were detected by enhanced chemiluminescence detection procedure (Amersham Pharmacia Biotech, GE Healthcare, Velizy-Villacoublay, France) and quantified using QuantityOne (Bio-Rad). Equal loading was checked by reprobing the membrane with β-actin or α-tubulin antibody (Santa Cruz Biotechnologies).
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