The following antibodies were used to assess protein levels: anti-RhoA (Cell Signaling Technology), antiphosphate MYPT (EMD Millipore), antiphosphomyosin light chain (MLC; Cell Signaling Technology), anti-MLC (Cell Signaling Technology), and anti-β-actin (Sigma-Aldrich Co.). The secondary antibodies used were horseradish peroxidase-conjugated goat antirabbit and antimouse IgG (Santa Cruz Biotechnology Inc.). Protein concentrations were assayed using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories, Hercules, CA, USA). Immunoprecipitates were separated on SDS gels. Samples of total protein (50 µg) were separated using 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with primary antibody. Immunocomplexes were detected using the appropriate horseradish peroxidase-conjugated secondary antibody and observed using a UVP AutoChemi Image and Analysis System (UVP, Upland, CA, USA) with enhanced chemiluminescence (Amersham-Pharmacia International, Piscataway, NJ, USA).
Anti mlc
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-MLC is a laboratory reagent that specifically binds to and detects myosin light chain (MLC) proteins. MLC is a component of the myosin enzyme complex, which is involved in cellular processes such as muscle contraction and cell motility. The Anti-MLC product can be used to identify and quantify MLC in various biological samples through techniques like Western blotting, immunohistochemistry, and flow cytometry.
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Lab products found in correlation
Anti pmlc, by Cell Signaling Technology (5 mentions)
Anti phospho mlc, by Cell Signaling Technology (4 mentions)
Anti rhoa, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti p cofilin, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Uvp autochemi image and analysis system, by Analytik Jena (1 mentions)
Anti p erk, by Santa Cruz Biotechnology (1 mentions)
Anti raf, by Abcam (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Anti erk, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti mypt1, by Cell Signaling Technology (1 mentions)
Anti p mypt1, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti trka, by Cell Signaling Technology (1 mentions)
Anti na k atpase, by Abcam (1 mentions)
Pretroq, by Takara Bio (1 mentions)
13 protocols using anti mlc
1
Immunoblotting for Protein Quantification
2
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.
3
Linx Protein Expression and Antibody Analysis
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cDNA encoding mouse Linx (clone 6826287, GenBank accession number BC096531) was purchased from Open Biosystems and subcloned into pcDNA3.1 (Invitrogen) and pRetroQ (Clontech) vectors to fuse Linx with the V5 and SF tag, respectively. GFP-Rho-kinase cDNA was provided by M. Amano and K. Kaibuchi (Nagoya University). The following antibodies were used in this study; anti-Linx (Islr2; R&D Systems), anti-Linx (Islr2; Abnova), anti-Ret51 (IBL, Gumma, Japan), β-actin (Sigma), anti-Tau-1 (Millipore), anti-TrkA (Cell Signaling Technology), anti-phospho-MLC (Ser19; Cell Signaling Technology), anti-MLC (Cell Signaling Technology), anti-Rho-kinase 2 (ROCK2, Abcam), anti-L1 (Millipore), anti-E-cadherin (Cell Signaling Technology), anti-Na+/K+-ATPase (Abcam), and anti-GFP (MBL, Nagoya, Japan).
4
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.
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.
5
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.
6
Protein Expression Analysis in Vascular Cells
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Proteins were extracted from either blood vessels or VSMCs. The levels of protein expression or phosphorylation were measured using Western blot as described previously [5 (link)]. The immunoblots were probed with anti-p-myosin light chain (#3671, Cell Signaling Technology), anti-MLC (#3672, Cell Signaling Technology), anti-Bcl-xL (#2764, Cell Signaling Technology) and anti-β-actin antibodies (#4970, Cell Signaling Technology) overnight at 4°C followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were visualized with ECL-plus reagent.
7
Immunoblotting Analysis of Endothelial Tight Junctions
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bEnd.3 cells were lysed on ice in RIPA buffer. After protein content determination by the BCA protein assay kit, the samples were heat denatured, resolved by 12% SDS–PAGE, and transferred onto nitrocellulose blotting (NC) membranes. The membranes were probed with rabbit anti‐Claudin‐5, anti‐ZO‐1, anti‐MLCK (1:2500, LSBio), anti‐pMLC (1:1000, Cell Signaling Technology, Danvers, MA, USA) and anti‐MLC (1:1000, Cell Signalling Technology), followed by HRP labeled anti‐rabbit or anti‐mouse secondary antibodies (1:100, Vector). Protein signals were detected by enhanced chemiluminescence (Pierce), with β‐actin used as an internal control.
8
Western Blot Analysis of Tight Junction Proteins
9
Antibody-based Protein Analysis
10
Endothelial Barrier Regulation Protocols
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ADM, histamine, 150 kDa FITC-dextran and Y27632 were purchased from Sigma-Aldrich (Toluca, Mexico). The following antibodies have been used: monoclonal anti-cortactin (clone 4F11, 1:3000, Millipore, Naucalpan, Mexico); anti-γ-tubulin (clone GTU-88, 1:5000) and polyclonal anti-MLCK (SAB1300116, 1:500) (Sigma-Aldrich), monoclonal anti-PECAM-1 (clone MEC13.3., 1:400, BD, Franklin Lakes, NJ); polyclonal anti-ROCK1 (#4035, 1:1000), anti-MLC (#3672, 1:1000), anti-pMLC (#3671, Ser-19, 1:1000), anti-pMLC (#3674, Thr-18/Ser-19, 1:1000), anti-mDia1 (clone 51/mDia1, 1:1000), anti-ezrin (#3145, 1:1000), anti-p-ezrin (#3149, 1:1000), anti-cofilin (#3312S, 1:1000) and anti-p-cofilin (#3313P, 1:1000) (Cell Signaling, Danvers, MA), anti-VE-cadherin (C19, sc6458, 1:400) and species-specific peroxidase-labelled secondary antibodies (1:5000, Santa Cruz Biotechnologies, Santa Cruz, CA). Alexa-568-phalloidin (1:100, A12380; and species-specific Alexa-labelled secondary antibodies (1:2000) were from Invitrogen (Carlsbad, CA).
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