Primary antibodies to the following proteins were used: YAP, phospho-YAP Ser127, PLK1 (208G4), MST2, phospho-MLC, and MLC (Cell Signaling Technology Inc.); LATS1 and LATS2 (Bethyl Laboratories Inc.); YAP, ECT2, tubulin, GFP, and PATJ (Abcam); YAP (H215), YAP (63.7), Anillin (H-300), RHOA (119), RHOA (26C4), ECT2 (H300), ECT2 (C-20), and Centrin-2 (N-17)-R (Santa Cruz Biotechnology); RacGAP1, YAP1, PLK1, Flag M2, and Flag M5 (Sigma-Aldrich); Cep55 (Abnova); and PATJ (Novus). Secondary antibodies used included Alexa Fluor goat anti-rabbit 488 and 568, goat anti-mouse 488 and 568, donkey anti-goat 488 and 568 (Invitrogen) or goat anti-mouse immunoglobulin G (IgG)–horseradish peroxidase (HRP), and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology). DAPI (Sigma-Aldrich) was used to stain DNA/nuclei.
Phospho mlc
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-MLC is a laboratory equipment product that detects and quantifies phosphorylated myosin light chain (MLC) proteins. It is used to study cellular signaling pathways and processes involving MLC phosphorylation.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt, by Cell Signaling Technology (4 mentions)
Phospho mypt, by Cell Signaling Technology (3 mentions)
Ve cadherin, by Santa Cruz Biotechnology (3 mentions)
Phospho erk, by Cell Signaling Technology (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Gm130, by BD (2 mentions)
Gapdh, by Proteintech (2 mentions)
α tubulin, by Merck Group (2 mentions)
Phospho mypt1, by Cell Signaling Technology (2 mentions)
Phospho ampk, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Lats1, by Fortis Life Sciences (1 mentions)
Lats2, by Fortis Life Sciences (1 mentions)
Goat anti mouse immunoglobulin g igg horseradish peroxidase hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Phospho yap ser127, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Flag m2, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Clone 138g6, by Cell Signaling Technology (1 mentions)
17 protocols using phospho mlc
1
Comprehensive Antibody Panel for Cytoskeletal and Signaling Proteins
2
Western Blotting Protein Analysis
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We lysed tissues in cold RIPA buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with complete miniprotease and phosphatase inhibitor cocktail (Pierce, Rockford, IL). We incubated lysates at 4°C with gentle rocking for 30 min, sonicated on ice for 30 s (in 5 s bursts) and then centrifuged at 12,800 rpm for 15 min at 4°C. We determined protein concentration by Bradford assay (Bio-Rad, Hercules, CA). We separated 20 μg of protein by SDS-PAGE on 7.5% resolving gels (Bio-Rad) and transblotted onto polyvinylidene fluoride membranes (Millipore). We incubated the membranes with a 1:1,000 dilution of antibodies against Akt (catalog 9272, Cell Signaling), phospho-Akt Ser473 (clone 193H12, Cell Signaling), MLC (catalog 3672S Cell Signaling) phospho-MLC (clone 519, Cell Signaling), MYPT (catalog 2634S, Cell Signaling) phospho-MYPT (catalog 5163, Cell Signaling), RhoA (clone 67139, Cell Signaling), PTEN (clone 138G6, Cell Signaling), or GAPDH (clone 14C10, Cell Signaling) followed by a secondary HRP-conjugated antibody. For evaluation of total Akt, MLC or MYPT we stripped and reprobed membranes that had been blotted for phospho-versions of these proteins. Blots were developed using the enhance chemical luminescence system (Amersham).
3
Immunoblotting and Immunofluorescence Assay Protocol
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Blebbistatin (B05060), low-endotoxin BSA (A8806), PA (P9767), and Y27632 (Y0503) were from Sigma Aldrich (St. Louis, MO), while cis-PO/palmitoleic acid (10009871) was from Cayman Chemicals (Ann Arbor, MI). Antibodies used for immunoblotting were α-actinin (Millipore Sigma; A7811), β-actin (Cell Signaling; 4790), α-catenin (ThermoFisher; 13-9700), β-catenin (BD Biosciences; 610153), VE-cadherin (Santa Cruz; 9989), RhoA (Santa Cruz; c-418), IRDye 800CW goat anti-rabbit immunoglobulin G (IgG) (LI-COR; 9253211), and IRDye 680LT goat anti-mouse IgG (92668070). Antibodies and reagents used for immunofluorescence were α-catenin (ThermoFisher; 13-9700), β-catenin (BD Biosciences; 610153), DAPI (4’,6-Diamidino-2-phenylindole, dihydrochloride; ThermoFisher; D1306), GM130 (BD Biosciences; 610823), phospho-MLC (Cell Signaling; 3674), phalloidin (Alexa Fluor 555) (ThermoFisher; A34055), phospho-YAP S127 (Abcam; 76252), YAP (Cell Signaling 14074), VE-cadherin (Santa Cruz; 9989), goat anti-rabbit IgG secondary antibody (Alexa Fluor 488) (ThermoFisher; A11008), goat anti-mouse IgG secondary antibody (Alexa Fluor 647) (ThermoFisher; A21235). Dako fluorescence mounting medium (S3023) was from Aligent. Y227632 (Y0503) was from Millipore Sigma (Burlington, MA). The plasmid encoding GFP–β-actin was kindly provided by Sergio Grinstein (Hospital for Sick Children, Toronto, Ontario, Canada).
4
Rho Pathway Regulation of TRPV4 Signaling
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TRPV4, ROCK1, ROCK2, and paxillin (PXN) antibodies were purchased from Abcam (Cambridge, MA, USA). RhoA, RhoB, RhoC, CDC42, RAC1/2, LIMK, phospho-LIMK, cofilin, phospho-cofilin, MLC, phospho-MLC, MYPT, and phospho-MYPT antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). GAPDH were purchased from Proteintech (Rosemont, IL, USA). Rho pathway antagonist Y27632, calcium chelator BAPTA-AM, TRPV4 agonist GSK1016790A, and its antagonist HC067047 were purchased from Selleck (Shanghai, China). Opti-MEM medium and Lipofectamine RNAiMAX reagent, Fluo-4 AM, Rhodamine Phalloidin, puromycin, and DAPI were purchased from Invitrogen (Massachusetts, USA).
5
Antibodies for Cellular Localization
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Generation of pericentrin antibodies and Borg5 rabbit and chicken antibodies were described previously (Liu and Zheng, 2009 (link); Vong et al., 2010 (link)). All the other antibodies and molecules used in this study were commercially available, including α-tubulin (T9026, 1:1000; Sigma-Aldrich), actin (A3853, 1:200; Sigma-Aldrich), GM130 (610822, 1:200; BD Biosciences, Franklin Lakes, NJ), isolectin β4 (I21412, 1:200; Invitrogen), Nkx2.5 (sc-8697, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA), PECAM-1 (550274, 1:10; BD Biosciences), phalloidin (A22287 or A12379, 1:200; Invitrogen), phospho-MLC (3674, 1:200; Cell Signaling Technology, Danvers, MA), Sept7 (18991, 1:1000; IBL, Minneapolis, MN), and VE-cadherin (sc-6458, 1:50; Santa Cruz Biotechnology).
6
Western Blot Analysis of Signaling Pathways
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HuSMCs were lysed in TNE buffer (10 mM Tris, pH 7.8, 1% Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, protease inhibitor cocktail (Sigma) and phosphatase inhibitors (Sigma)) for 30 minutes at 4°C. 100 µg of total cell lysate protein was loaded on SDS-polyacrylamide gel and proteins were resolved and transferred to PVDF membrane. The following primary antibodies were used: TrkB (Cat# 07–225, Millipore), AKT (Cat# 46915, Cell Signaling Technology), phospho-AKT (Cat# 92715, Cell Signaling Technology), ERK (Cat# 46955, Cell Signaling Technology), phospho-ERK (Cat# 57265, Cell Signaling Technology), MLC (Cat# 3672, Cell Signaling Technology), and phospho-MLC (Cat# 3674, Cell Signaling Technology) antibodies. Anti β-actin (Cat# A-5441, Sigma) was used as a loading control. The respective secondary antibodies (anti-rabbit or anti-mouse IgG) were conjugated with HRP. Chemiluminescence of the blots was detected using ECL Advance Western Blotting Detection kit.
7
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.
8
Porcine Collagen and TGF-β1 Signaling
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Native porcine type I collagen was provided by Nitta Gelatin (Osaka, Japan), and fetal bovine serum (FBS) and Eagle’s minimum essential medium (MEM) were obtained from Invitrogen-Gibco (Gibco). R&D Systems provided recombinant human TGF-β1 (Minneapolis, MN). Antibodies against Smad2/3, phospho-Smad2/3, ERK, p38, phospho-p-38, Akt, phospho-Akt, myosin light chain (MLC), and phospho-MLC were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were provided by Proteintech (Rosemont, IL). Antibodies against α-SMA and SFN were provided by Sigma-Aldrich. Fibronectin was purchased from Abcam (Boston, MA). Beyotime (Shanghai, China) provided bovine serum albumin (BSA), enhanced chemiluminescence (ECL) reagents, horseradish peroxidase (HRP), and secondary antibodies. 4ʹ,6-diamidino-2-phenylindole (DAPI) was purchased from MP Biomedicals (Irvine, CA), and rhodamine phalloidin was obtained from Cytoskeleton (Denver, CO). Cell Counting Kit-8 (CCK-8) was bought from Dojindo Molecular Technology (Kumamoto, Japan).
9
Immunoblotting and Immunostaining for Podocyte Injury
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Antibodies against phospho-cofilin, total-myosin light-chains (MLC), α-tubulin, LIMK1, and phospho-MLC were purchased from Cell Signaling (Beverly, MA, USA). Antibodies against NQO1 (for immunostaining), β-actin, zonula occludens-1 (ZO-1), nephrin, α-actinin-4, Wilms’ tumor 1 (WT1), CD2-associated protein (CD2AP), podocin, gp91phox (NOX2), NOX4, gp67phox, gp22phox, and total-cofilin were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against synaptopodin were purchased from Novus Biologicals (for western blot, Centennial, CO, USA) or Progen (for immunostaining, Wayne, PA, USA). Alexa 546 anti-rabbit and Alexa 488 anti-mouse IgG were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Streptozotocin (STZ) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human NQO1 protein was obtained from R&D systems (MN, USA).
10
Collagen Invasion Assay with IGF-1
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Y-27632 (Tocris), geodin (Santa-Cruz), blocking human IGF-1 antibody (R&D Systems, AF-291-NA) and recombinant IGF-1 (Sigma-Aldrich, I1146) were used in collagen invasion assay. The following antibodies: E-cadherin (Cell Signaling, #3195), phospho-p120 catenin (Tyr228) (Cell Signaling, #2911), PAI-1 (Cell Signaling, #11907), phospho-ERM (Cell Signaling, #3141), MLC (Cell Signaling, #3672), phospho-MLC (Cell Signaling, #3674), α-SMA (Abcam, ab5694), α-tubulin (Sigma-Aldrich, T5168), β-actin (Sigma-Aldrich, A5441) were used for immunoblotting or immunofluorescence on frozen sections. For in vivo experiments, Y27632 and PQ401 were obtained from AbMole Biosciences.
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