Whole cell extracts were prepared and transferred to membranes with standard methods as described before [15] (link). Membranes were incubated overnight at 4°C with the following antibodies: anti-PDCD10 (#SAB1305161), anti-β-Actin (clone AC-74, #A2228) from Sigma-Aldrich; anti-phospho cofilin (sc-12912), anti-cofilin (sc-33779) and anti- Cyclin D1 (sc-8396) from Santa Cruz (Dallas, TX, USA); anti-phospho-Mypt1 (#5163), anti-Mypt1 (#2634), anti-phospho-MLC2 (#3671), anti-MLC2 (#8505) and anti-PARP (#9542) from Cell Signaling Technology (Danvers, MA, USA); anti-cleaved Caspase 3 (2305-PC-100) from Trevigen (Gaithersburg, MD, USA). GTP-bound Rho was assayed with Rho assay reagent according to manifacturer instructions (#17-294; Millipore, Burlington, MA, USA). Chemio-luminescence was detected with Alliance Mini WL2M system (Uvitec, Cambridge, UK).
Anti phospho mlc2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-MLC2 is a primary antibody that detects phosphorylated myosin light chain 2 (MLC2) in Western blotting applications. MLC2 phosphorylation is a key regulatory mechanism for myosin II activity.
Automatically generated - may contain errors
Lab products found in correlation
Anti mlc2, by Cell Signaling Technology (6 mentions)
Anti phospho mypt1, by Cell Signaling Technology (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti β actin clone ac 74, by Merck Group (1 mentions)
Rho assay reagent, by Merck Group (1 mentions)
Alliance mini wl2m system, by Uvitec (1 mentions)
Anti pdcd10, by Merck Group (1 mentions)
Anti cyclin d1 sc 8396, by Santa Cruz Biotechnology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Anti phospho cpi 17, by Santa Cruz Biotechnology (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Anti lamin a c, by Cell Signaling Technology (1 mentions)
Anti smad2 3, by Cell Signaling Technology (1 mentions)
Anti caveolin 1, by Abcam (1 mentions)
Anti col1a2, by Abcam (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Recombinant human tgf β2, by Fujifilm (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Anti phospho smad2, by Cell Signaling Technology (1 mentions)
8 protocols using anti phospho mlc2
1
Western Blot Analysis of Cellular Signaling
2
Protein Kinase C Signaling Assay
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Then, 15 h post transfection, cells were treated with either 1 µM PMA (a PKC agonist, Sigma-Aldrich, St Louis, MO, USA) or 0.1% DMSO for 3 h, then washed in PBS, and lysed in RIPA buffer. Cleared lysates were run on 12% SDS-PAGE and blotted with anti-GFP (Santa Cruz B2, Santa Cruz, CA, USA), anti-phospho-Cpi-17 (Santa Cruz-17560, Santa Cruz, CA, USA), anti-Mlc2 (Cell Signaling #3672, Danvers, MA, USA), and anti phospho-Mlc2 (Cell Signaling #3675, Danvers, MA, USA).
3
Investigating Transforming Growth Factor-β2 Signaling
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Recombinant human TGF-β2 was purchased from Wako Pure Chemical Industries (Osaka, Japan). Y-27632 and SB203580 were purchased from Merck Millipore (Darmstadt, Germany). The anti-COL1A2 (1:2,000 dilution;), anti-Caveolin 1 (1:200 dilution), and anti-myocilin (1:100 dilution) antibodies were obtained from Abcam (Cambridge, UK). The anti-MLC2 (1:800 dilution), anti-phospho-MLC2 (1:700 dilution; Thr18/Ser19), anti-Smad2/3 (1:1,000 dilution), anti-phospho-Smad2 (1:1,000 dilution; Ser465/467), anti-α-tubulin (1:1,000 dilution), and anti-lamin A/C (1:2,000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, MA). The anti-β-actin mouse monoclonal antibody (1:10,000 dilution;) was obtained from Sigma (St. Louis, MO). The anti-collagen 4 α 5 chain (1:50 dilution) and anti-matrix protein gla (1:400 dilution) antibodies were purchased from Santa Cruz (Dallas, TX). The tissue inhibitor of matrix metalloproteinase 3 antibody (1:1,000 dilution) was obtained from Thermo Pierce (Rockford, IL). The anti-vascular cell adhesion protein 1 antibody (1:100 dilution) was obtained from R&D systems (Minneapolis, MN).
4
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.
5
Protein Expression Analysis by Western Blot
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The tissue was homogenized in radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Biotechnology, Shanghai, China) and incubated on ice for 30 min. The supernatant following centrifugation (at 15,000 × g for 15 min at 4°C) was used for western blotting. Western blotting was performed as described previously (25 (link)). Blots were incubated overnight with the following primary antibodies: Anti-podoplanin (rabbit polyclonal antibody; 1:300; cat. no. 251419; Abbiotec, San Diego, CA, USA), anti-β-tubulin (mouse monoclonal antibody; 1:5,000; cat. no. AT0003; CMCTAG, Inc., Milwaukee, WI, USA) and anti-cofilin (rabbit monoclonal antibody; 1:1,000; cat. no. 5175, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-cofilin (rabbit monoclonal antibody; 1:1,000; cat. no. 3313; Cell Signaling Technology), anti-ERM (rabbit monoclonal antibody; 1:1,000; cat. no. 3142; Cell Signaling Technology), anti-phospho-ERM (rabbit monoclonal antibody; 1:1,000; cat. no. 3726; Cell Signaling Technology), anti-MLC-2 (rabbit monoclonal antibody; 1:1,000; cat. no. 8505; Cell Signaling Technology) and anti-phospho-MLC-2 (1:1,000; cat. no. 3671; Cell Signaling Technology). Western blots were visualized using an electrophoretic gel imaging system (Shanghai, China).
6
Protein Expression Analysis by Western Blot
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Protein extracts from cells were prepared using 2% SDS lysis buffer including protein phosphatase inhibitor (Thermo Scientific™, #78,440). Total protein (20 μg) was subjected to 10% (v/v) SDS-PAGE gels and transferred to nitrocellulose filter membrane (Pall, #P-N66485). After blocking with 5% bovine serum albumin (BSA) for 2 h at 37 ℃, the membranes of proteins with different molecular weight (KDa) were cut according to the location of the markers (GenStar, M222) with enough spaces left at the edges of the first and the last sample lanes, then the cropped membranes were incubated with primary antibodies at 4℃ overnight. The primary antibodies anti-KRT7 (1:1000; Proteintech, #17,513–1-AP), anti-E-cadherin (1:5000; Proteintech, #20,874–1-AP), anti-P-cadherin (1:1000; Proteintech, #13,773–1-AP), anti-RHOA (1:1000; Proteintech, #10,749–1-AP), anti-Phospho-MLC2 (1:1000; Cell Signaling Technology, #3671), anti-β-Actin (1:20,000; Proteintech, #66,009–1-lg) and anti-GAPDH (1:50,000; Proteintech, #60,004–1-lg) were used and then incubated with HRP-conjugated secondary antibodies (1:5000; Proteintech, #SA00001-1 and # SA00001-2) at room temperature for 1 h and imaged through ECL kit (Beyotime, #P0018AM).
7
MLC Phosphorylation Measurement in Endothelial Cells
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The myosin light chain (MLC) phosphorylation in human endothelial cells was measured by western blot analysis (Aslam et al., 2010 (link)). Therefore, cells were harvested in 2x SDS-PAGE sample buffer and separated by 12.5% SDS-PAGE and transferred onto nitrocellulose membranes by semi-dry blotting. Membranes were probed using anti-phospho-MLC-2 (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma-Aldrich) in Tris-buffered saline with 0.1% (v/v) Tween 20 and 5% (w/v) BSA in a dilution of 1:3000 and 1:5000, respectively. Respective secondary HRP-conjugated anti-rabbit and anti-mouse IgG antibodies (Amersham BioSciences Buckinghamshire, UK) were used in a dilution of 1:10:000. Immunoreactivity was detected by Fusion-FX7 (PeqLab, Erlangen, Germany) with enhanced chemiluminescence and quantified by densitometric analysis by using Quantity One software (Bio-Rad, Munich, Germany). MLC phosphorylation was expressed in relation to the intracellular amount of actin.
8
Human Renal Cell Carcinoma Protocol
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Cell culture and reagents. Human RCC 786-0 cells were obtained from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 medium containing penicillin (50 U/ml), streptomycin (50 U/ml) and 10% FbS according to the ATCC procedures. Media came from ATCC. Supplements and FbS were obtained from Gibco (Grand Island, NY, USA). Honokiol 98% (HonoPure ® ) was provided by EcoNugenics, Inc. (Santa Rosa, CA, USA) and dissolved in DMSO at a concentration of 80 mM then stored at -20˚C. Rho-kinase inhibitor Y-27632 was purchased from Calbiochem (Darmstadt, Germany). Rhodamine phalloidin was purchased from Molecular Probes (Grand Island, NY, USA). Methanol-free formaldehyde solution 16% was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). DMSO and other reagents were purchased from Sigma (St. Louis, MO, USA). Anti-RhoA, anti-phospho-RhoA and anti-β-actin antibodies were obtained from Santa Cruz biotechnology, Inc. (Santa Cruz, CA, USA). Anti-MLC2 and anti-phospho-MLC2 antibodies were obtained from Cell Signaling Technology, Inc. (beverly, MA, USA).
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