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Smooth muscle actin

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Smooth muscle actin is a type of actin protein found in the contractile fibers of smooth muscle cells. It is a key component of the cellular cytoskeleton and plays a crucial role in the regulation of muscle contraction and cell motility.

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Lab products found in correlation

Eclipse e800 microscope, by Nikon (3 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) E cadherin, by BD (2 mentions) Notch3 and hif 1α, by Abcam (2 mentions) Hey l, by LifeSpan BioSciences (2 mentions) Rbpjk, by Santa Cruz Biotechnology (2 mentions) Alexa fluor conjugated antibody, by Jackson ImmunoResearch (2 mentions) Von willebrand factor, by Agilent Technologies (2 mentions) Filamin a, by Abcam (1 mentions) Hyaluronan binding protein, by Merck Group (1 mentions) Ve cadherin, by R&D Systems (1 mentions) 3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions) Abc reagent, by Vector Laboratories (1 mentions) Antigen unmasking solution, by Vector Laboratories (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Biotinylated secondary antibody, by Vector Laboratories (1 mentions) Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions) Pecam cd31, by BD (1 mentions) Ve cad, by R&D Systems (1 mentions) Estrogen receptor, by Abcam (1 mentions)

21 protocols using smooth muscle actin

1

Pulmonary Vessel Remodeling Histological Analysis

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Lung histology was examined by elastic Van Gieson, and hematoxylin and eosin staining. Peripheral vessels <50 μm diameter were counted at ×40 magnification and pulmonary vascular remodeling was expressed as the proportion of vessels with double elastic lamina (>75% of the circumference) to total vessels counted.
For immunohistochemistry examination, lung sections were stained for smooth muscle actin (1/200; Sigma), Von Willebrand factor (1/100; Dako), CD68 (1/200; Serotec), β-catenin (1/100; Abcam), or nuclear factor of activated T cells 1 (NFAT-1; 1/50, Novus Biological), with appropriate horseradish peroxidase-conjugated secondary antibodies (1/200). We counted macrophages (CD 68 + ) as previously described. 16 (link) For double immunofluorescence, sections were incubated with glucose transporter 1 (GLUT1; 1/50, Abcam), carbonic anhydrase (1/100; Novus Biological), and smooth muscle actin (1/200; Sigma), detected with secondary antibodies, Alexa 488 antimouse, and Alexa 568 antirabbit (1/1000; Invitrogen), with mounting solution containing 4′,6-diamidino-2-phenylindole. Images were obtained with a Leica laser confocal microscope (TCS SP2 AOBS).
Cotroneo E., Ashek A., Wang L., Wharton J., Dubois O., Bozorgi S., Busbridge M., Alavian K.N., Wilkins M.R, & Zhao L. (2015). Iron homeostasis and pulmonary hypertension: iron deficiency leads to pulmonary vascular remodeling in the rat. Circulation research, 116(10).
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2

Immunofluorescence Antibody Labeling Protocol

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Antibodies used for cell or embryo immunofluorescence were used at a dilution of 1/100 unless otherwise specified. Antibodies were raised against Isl1 (Developmental Hybridoma bank, Iowa University 1/50), Tbx20 (novus biological H00057057-B01), Msx1 (Abcam ab93287), Tbx3 (Santa Cruz sc-178721), aggrecan (Chemicon AB1031), versican (Chemicon AB1033), smooth muscle actin (Sigma A-2547), Filamin A (Epitomics, CA, USA), vimentin dylight 550 (Novus biological, VM452, 1/200), periostin (Abcam ab140141), CD31 (BD pharmigen, WM59), VE-cadherin (R&D systems/MAB9381), Sox9 (a gift from Pr Wegner, University of Nurnberg, and Santa-Cruz Sc17431), GATA5 (Abcam ab11877), Hyaluronan-binding protein (Millipore 385911), collagen I (Abcam 34710), anti-Patched1 (Merck-Millipore, France, 06–1102), human Lamin A/C (Novacastra, NCL-LAM-A/C), and anti-NFATc (Santa-Cruz 17844 H10).
Neri T., Hiriart E., van Vliet P.P., Faure E., Norris R.A., Farhat B., Jagla B., Lefrancois J., Sugi Y., Moore-Morris T., Zaffran S., Faustino R.S., Zambon A.C., Desvignes J.P., Salgado D., Levine R.A., de la Pompa J.L., Terzic A., Evans S.M., Markwald R, & Pucéat M. (2019). Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis. Nature Communications, 10, 1929.
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3

Immunohistochemical Analysis of Cellular Markers

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Immunohistochemistry was performed as previously described using antibodies for E-cadherin (Cell Signaling), smooth muscle actin (Sigma), p63 (Santa Cruz Biotechnology), Ki67 (Vector Laboratory, VP-K452) or p21 (Santa Cruz Biotechnology, sc-397) (31 (link)). Slides were dewaxed, rehydrated with a graded alcohol series, antigen retrieval was performed with antigen unmasking solution (Vector Labs), and endogenous peroxidases were quenched by incubation in H2O2. Slides were blocked with 2.5% serum, incubated with primary antibody overnight, washed, and incubated with biotinylated secondary antibody (Vector Labs) followed by incubation with ABC reagent (Vector Labs), and color development with DAB (Vector Labs). Slides were counterstained with haematoxylin, dehydrated, and mounted. TUNEL assays were performed as specified by the manufacturer using the DeadEnd Colormetric TUNEL assay system (Promega) and were counterstained with haematoxylin. Slides were imaged with a 20x objective on a Leica DMLB microscope and acquired using QCapture Software (QImaging Software). Labeling indices were calculated by blinded individuals who counted the number of positive cells (Ki-67, p21, TUNEL) and the total number of cells. Data are presented as the percentage of positive cells.
Powers G.L., Hammer K.D., Domenech M., Frantskevich K., Malinowski R.L., Bushman W., Beebe D.J, & Marker P.C. (2014). Phosphodiesterase 4D Inhibitors Limit Prostate Cancer Growth Potential. Molecular cancer research : MCR, 13(1), 149-160.
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4

Immunostaining Protocol for Cardiac Tissues

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Immunostaining was performed as previously described.29 (link) Briefly, hearts were collected in phosphate-buffered saline (PBS) on ice and then fixed in 4% paraformaldehyde at 4°C for 1 h. After washing in PBS, tissues were incubated in PBS/30% sucrose overnight at 4°C and embedded in optimum cutting tissue (OCT, Sakura) and snap frozen the following day. Cyrosections of 10 μm thickness were collected on positively charged slides. Tissues were blocked with PBS/0.1% Triton X-100/5% normal donkey serum (Jackson ImmunoResearch) for 1 h at room temperature, followed by primary antibody incubation overnight at 4°C. Signals were visualized with Alexa fluorescence-conjugated secondary antibodies (Invitrogen). For weak signals, we used horseradish peroxidase- or biotin-conjugated secondary antibodies and a tyramide signal amplification kit (PerkinElmer). Antibodies used were as follows: PECAM/CD31 (BD Pharmingen), Smooth muscle actin (Sigma), RFP (Rockland), GFP (Invitrogen), VE-CAD (R&D), and Estrogen receptor (Abcam). Images were acquired using an Olympus confocal microscope (FV1000), a Zeiss confocal microscope (LSM510), or a Leica M165 FC stereo microscope. The quantification of all experiments was performed by an observer blinded to the experimental design.
He L., Liu Q., Hu T., Huang X., Zhang H., Tian X., Yan Y., Wang L., Huang Y., Miquerol L., Wythe J.D, & Zhou B. (2016). Genetic lineage tracing discloses arteriogenesis as the main mechanism for collateral growth in the mouse heart. Cardiovascular Research, 109(3), 419-430.
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5

Immunohistochemistry for Angiogenesis Analysis

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Detailed description for tissue preparation and immunohistochemical staining for angiogenesis has been previously described14 (link). Formalin fixed tissue sections were incubated with antibodies against porcine endothelial marker CD-31 (R&D Systems, Minneapolis, MN) and smooth muscle actin (Sigma-Aldrich) followed by appropriate Alexa-Flour conjugated antibody (Jackson ImmunoResearch, West Grove, PA). Images were captured at 20X magnification with a Nikon E800 Eclipse microscope (Nikon, Tokyo, Japan) at the same exposure in 3 random fields. Capillaries were defined as structures 5-25 μm2 in a cross-sectional area. Arterioles were defined by localization of smooth muscle actin and CD31 staining. Arteriolar and capillary density was measured using Image J software (National Institutes of Health, Bathesda, MD) in a blinded fashion. Representative images have been included in this manuscript.
Sabe A.A., Potz B.A., Elmadhun N.Y., Liu Y., Feng J., Abid M.R., Abbott J.D., Senger D.R, & Sellke F.W. (2015). Calpain Inhibition Improves Collateral Dependent Perfusion in a Hypercholesterolemic Swine Model of Chronic Myocardial Ischemia. The Journal of thoracic and cardiovascular surgery, 151(1), 245-252.
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6

Western Blot Analysis of Smooth Muscle Actin

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Western blotting was done as described previously in polyacrylamide gradient gels (8–16%; Invitrogen Corporation) run in tris glycine buffer under reduced conditions (Wang et al. 2007). The primary antibodies were smooth muscle actin (A2547; Sigma‐Aldrich, St. Louis, MO) and GAPDH (ab8245; Abcam, Cambridge, MA) The images were quantified, using NIH ImageJ software.
Correll K.A., Edeen K.E., Redente E.F., Zemans R.L., Edelman B.L., Danhorn T., Curran‐Everett D., Mikels‐Vigdal A, & Mason R.J. (2018). TGF beta inhibits HGF, FGF7, and FGF10 expression in normal and IPF lung fibroblasts. Physiological Reports, 6(16), e13794.
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7

Comprehensive Antibody Panel for Cellular Signaling

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The following antibodies were used in this study. PAK1 (2602), p-PAK1/2 (2605), p-AKT (3787), p-ERK (4370), ERK (9102), p-MEK1/2 (9121), MEK1/2 (9122), β-Catenin (8480), cleaved caspase-3 (9661) (Cell Signaling Technology), AR (N-20, SC-816), p63 (4A4), Smooth Muscle actin (A5228), tubulin, vinculin, actin (Sigma-Aldrich), ERG (abcam 92513)
Gao X., Wang Y., Ribeiro C.F., Manokaran C., Chang H., Von T., Rodrigues S., Cizmecioglu O., Jia S., Korpal M., Korn J.M., Wang Z., Schmit F., Jiang L., Pagliarini R., Yang Y., Sethi I., Signoretti S., Yuan G.C., Loda M., Zhao J.J, & Roberts T.M. (2022). Blocking PI3K p110β Attenuates Development of PTEN-Deficient Castration-Resistant Prostate Cancer. Molecular cancer research : MCR, 20(5), 673-685.
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8

Antibody Staining for Cell Markers

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Antibodies to NMHC II-A (Covance, 1:1000) II-B (Covance, 1:3000) and II-C (Covance, 1:2000) were used for immunofluorescence staining. Antibodies to E-cadherin (BD Transduction Labs, 1:250), N-cadherin (Zymed, 1:250), p21 (BD Pharmingen, 1:50), TP53 (Leica NCL, 1:1000), smooth muscle actin (Sigma, 1:1000), and vimentin (Sigma, 1:40) were used at the dilutions indicated.
Anne Conti M., Saleh A.D., Brinster L.R., Cheng H., Chen Z., Cornelius S., Liu C., Ma X., Van Waes C, & Adelstein R.S. (2015). Conditional deletion of nonmuscle myosin II-A in mouse tongue epithelium results in squamous cell carcinoma. Scientific Reports, 5, 14068.
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9

Prostate Cancer Immunohistochemistry and IF

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Mouse prostatic tissues were dissected from cPten−/− Luc males. After overnight fixation in 4% PFA, tissues were processed and sectioned. IHC was performed as previously described with anti-mouse AR antibody (Santa Cruz) at 1:1000 dilution (Liao et al. 2010a (link)). For IF staining, mouse NPF and CAF cells were seeded on Lab Tek II chamber slides (Thermo Scientific) with BFS medium for 24 hours, followed by fixing in 4% PFA for 10 mins and washing with PBS. After blocking, Smooth Muscle Actin (SMA) antibody conjugated with Cy3 dye (Sigma) was applied for 1 hour at a 1:60 dilution. For vimentin (Santa Cruz; 1:50 dilution) and N-Cadherin (Santa Cruz; 1:50 dilution), the primary antibody was applied for 1 hour, following by the staining of secondary antibody conjugated with either FITC or rhodamine. Samples were counter-stained with DAPI and then examined with fluorescent microscopy (Observer.Z1, ZEISS).
Liao C.P., Chen L.Y., Luethy A., Kim Y., Kani K., MacLeod A.R, & Gross M.E. (2017). Androgen Receptor in Cancer-Associated Fibroblasts Influences Stemness in Cancer Cells. Endocrine-related cancer, 24(4), 157-170.
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10

Immunohistochemical Analysis of Tissue Samples

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Immunohistochemical analysis was performed on 4-µm formalin-fixed paraffin-embedded (FFPE) tissue sections using the following antibodies and conditions: S-100 protein (Dako, Carpinteria, CA; polyclonal; 1:3000), smooth muscle actin (SMA; Sigma, St. Louis, MO; clone 1A4; 1:20,000), desmin (Sigma; clone D33; 1:500), myogenin (MYF4) (Novocastra, Newcastle, U.K.; Clone LO26; 1:600), EMA (Dako; clone E29; 1:200), TLE1 (Santa Cruz, Santa Cruz, CA; polyclonal; 1:400) and beta-catenin (Novocastra; clone 17C2; 1:50). The Envision Plus detection system (Dako) was used for all antibodies. Appropriate positive and negative controls were used throughout.
Wong W.J., Lauria A., Hornick J.L., Xiao S., Fletcher J.A, & Marino-Enriquez A. (2015). Alternate PAX3-FOXO1 Oncogenic Fusion in Biphenotypic Sinonasal Sarcoma. Genes, chromosomes & cancer, 55(1), 25-29.
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