The following antibodies were used: Alpha Smooth Muscle Actin (α‐SMA) (1:1000; Sigma A2547), Periostin (1:100; Santa Cruz, SC67233), collagen I (1:500; MD Bioproducts, #203002A), phospho‐Akt (1:500; Cell Signaling, #4058S), Akt (1:500; Cell Signaling, #9272), pSmad2 (1:500; Cell Signaling, #3108S), Smad2/3 (1:500; Cell Signaling, #5678S), β‐actin (1:5000; Sigma, #A5441), and SPRR3 (1:500; Proteintech group 11742‐1‐AP, or custom made) p‐p38 (1:500; Cell Signaling), p38 (1:500; Cell Signaling), FAK (1:1000; Cell Signaling, #3285), p‐FAK (1:500; Cell Signaling; #3283), GAPDH (1:500; Millipore; MAB374), p‐PDGFRβ (Tyr751) (1:1000; Cell Signaling; #3161), PDGFRβ (28E1) (1:1000; Cell Signaling; #3169), integrin β1 (1:1000; R&D; #AF2405), total integrin β1 (flow cytometry, BD Pharmingen #550530), active integrin β1 (BD Pharmingen; #553715), and integrin β1 (Abcam; ab24693; 10 ug/ml for proliferation).
Alpha smooth muscle actin α sma
Manufactured by Merck Group
Sourced in United Kingdom, United States
Alpha Smooth Muscle Actin (α-SMA) is a cytoskeletal protein found in smooth muscle cells. It is a common marker used in cellular and molecular biology research to identify and characterize smooth muscle cells and myofibroblasts.
Automatically generated - may contain errors
Lab products found in correlation
Periostin, by Santa Cruz Biotechnology (3 mentions)
β actin, by Merck Group (3 mentions)
Smad2 3, by Cell Signaling Technology (2 mentions)
Sprr3, by Proteintech (2 mentions)
P pdgfrβ tyr751, by Cell Signaling Technology (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Integrin β1, by Abcam (2 mentions)
Integrin β1, by R&D Systems (2 mentions)
Gapdh, by Merck Group (2 mentions)
Total integrin β1, by BD (2 mentions)
Active integrin β1, by BD (2 mentions)
Pdgfrβ 28e1, by Cell Signaling Technology (2 mentions)
P smad2, by Cell Signaling Technology (2 mentions)
P fak, by Cell Signaling Technology (2 mentions)
Phospho histone h3, by Merck Group (2 mentions)
β catenin, by BD (2 mentions)
Protein block, by Agilent Technologies (1 mentions)
Reelin, by Abcam (1 mentions)
F4 80, by Abcam (1 mentions)
Von willebrand factor vwf, by Agilent Technologies (1 mentions)
12 protocols using alpha smooth muscle actin α sma
1
Comprehensive Antibody Analysis for Cell Signaling
2
Integrin and Signaling Pathway Analysis
3
Immunohistochemistry Analysis of Liver Tissue
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For immunohistochemistry, livers were fixed with formalin for 16 hours directly after harvest. Sections, 4 μm, were dewaxed and rehydrated, followed by antigen retrieval in boiling sodium citrate. Avidin and biotin were blocked according to the manufacturer's protocol (Vector, Peterborough, UK). Protein block (Dako, Cambridge, UK) or serum was added, and sections were then incubated with alpha‐smooth muscle actin (αSMA; Sigma‐Aldrich, Dorset, UK), collagen I, or GR1 (Ly6G) (Cambridge Bioscience, Cambridge, UK), F4/80 (Abcam, Cambridge, UK), or reelin (Abcam) antibodies at 4oC overnight and subsequent secondary antibody for 30 minutes. After ABC vector incubation, sections were incubated with 3,3'‐diaminobenzidine for 10 minutes and counterstained with hematoxylin. Picrosirius red (PSR) staining was performed as described.3 , 23 Thirty to 40 high‐power fields (magnification ×80) per section were randomly selected for each slide by an assessor blind to genotype. Images were analyzed in Photoshop CS3 for positively stained pixels and normalized to the total number of pixels. In the period acid–Schiff stain, the necrotic cell area was quantified in a blinded manner using the Image J Trainable Weka Segmentation tool.
4
Regorafenib and PDGF-BB Signaling
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Regorafenib (BAY 73-4506) and dimethyl sulfoxide (DMSO) were purchased from Selleckchem (Houston, TX, USA) and Sigma–Aldrich (St. Louis, MO, USA). Platelet-derived growth factor-beta (PDGF-BB) was purchased from R&D Systems (Minneapolis, MN, USA). Crystal violet and 4′,6′-diamidino-2-phenylindole (DAPI) were from Sigma–Aldrich (Munich, Germany). The following antibodies were used in this study: Phospho-ERK 1/2 (Thr 202/Tyr 204), ERK (Santa Cruz Biotechnology, Dallas, TX, USA), Phospho-SAPK/JNK (Thr183/Tyr185), SAPK/JNK (Cell Signaling, Danvers, MA, USA), von Willebrand factor (vWF, Dako, Hamburg, Germany), alpha smooth muscle actin (α-SMA, Sigma–Aldrich, Munich, Germany) and vinculin (Sigma–Aldrich, MO, USA).
5
Quantification of Carotid Artery Remodeling
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Carotid arteries were harvested on day 28 and fixed in 10% formaldehyde. Paraffin cross-sections were prepared at a thickness of 5 μm and stained with antibodies against CD31 (Abcam, Cambridge, UK), alpha smooth muscle Actin (αSMA, Sigma-Aldrich), hNrk (MyBioSource Inc.), mNrk (custom made, sequence: CLNNDPKSKKRQKAM) and calponin 1 (Santa Cruz Biotechnology, Dallas, Texas, USA) followed by the N-Histofine® MOUSESTAIN KIT (Nichirei Biosciences Inc., Tokyo, Japan) and Polink-2 Plus HRP anti Rabbit Detection Kit (GBI Labs, Mukilteo, WA, USA). At least three sets of sections at 150-μm intervals were used for morphometry of each arteries. Digitized images of H&E and elastic staining (Elastic Stain Kit, Sigma-Aldrich) were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, USA) to calculate the intimal area to the medial area ratio (I/M).
6
Immunohistochemistry of Cardiac Tissue
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Samples for immunohistochemistry were either fixed in PFA and paraffin-embedded or equilibrated through a sucrose series (to 15%) and subsequently mounted and frozen in OTC (Tissue-tek). In the latter case, air-dried sections were fixed with methanol or 4% PFA for immunostaining. Primary antibodies utilized were Scribble (Santa Cruz); Vangl2 (gift from Dr Charlotte Dean, London, UK), MF20 (DSHB), phospho-histone H3 (Millipore), cleaved caspase-3 (Cell Signalling), sarcomeric α actinin (Abcam), alpha-smooth muscle actin (α-SMA) (Sigma), cardiac troponin I (Hytest Ltd), Rac1 (Millipore), β-PIX (Millipore), β-catenin (BD Transduction Laboratories), N-cadherin (BD Transduction Laboratories), and connexin-43 (Chemicon). Alexa fluor 488 and 596-conjugated secondary antibodies (Invitrogen) were used to detect the primary antibody. Phalloidin (Sigma) was used to stain the actin cytoskeleton and wheat germ agglutinin (Alexa fluor 647; Invitrogen) was used to stain cell membranes. Cell nuclei were identified using DAPI. Immunofluorescence images were collected with using a Zeiss Axioimager Z1 fluorescence microscope equipped with Zeiss Apotome 2 (Zeiss, Germany). Acquired images were processed with the AxioVision Rel 4.9 software.
7
Protein Expression and Phosphorylation Analysis
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Cells were lysed in RIPA buffer in presence of protease and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein concentrations were determined by colorimetric assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific). Western blotting was performed using 35–40 µg of protein extracts (60 µg for the detection of LATS1 phosphorylation), using the following primary antibodies: α-tubulin (sc- 32293), Bcl-2 (sc-509) and Bcl-xL (sc-8392) were from Santa Cruz Biotechnology YAP (#12395), phosphorylated YAP (S127, #4911), LATS1 (#3477), phosphorylated LATS1 (#8654), MST2 (#3952), MOB1 (#13730), CTGF (connective tissue growth factor, also known as CCN2, #10,095), vinculin (#13901), pERK1/2 (#9106), ERK (#9102) and H3 (#4499) were from Cell Signaling (Danvers, MA, USA); β-actin (#A1978), alpha-smooth muscle actin, α-SMA (#A5228) was from Sigma-Aldrich; heat shock protein (HSP)72/73 (#HSP01) was from Calbiochem (San Diego, CA, USA). Enhanced Chemiluminescent Substrate method (LiteAblotTURBO, Euroclone) was used to detect immunostained bands, except for the detection of phosphorylated LATS1, by Clarity Max Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). ChemiDoc System instrument (Bio-Rad Laboratories) was used to acquire images, while ImageJ software was used for densitometric evaluation and normalization with relative controls.
8
Multi-Marker Imaging of Tumor Hypoxia
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Tumors and lungs were zinc fixed and paraffin-embedded. Tumor sections were stained using the Opal staining system and analyzed with InForm software using the phenotyping tool according to the manufacturer's instructions (PerkinElmer, Rodgau, Germany). Tumor sections were stained with the following antibodies: cleaved caspase (Cell Signaling, Cambridge, U.K.); Ki67 (abcam, Cambridge, U.K.); hypoxia-inducible factor 1-alpha (HIF1α) (Novus); panCytokeratin (abcam); CD31 (BD); alpha smooth muscle actin (αSMA) (Sigma-Aldrich); spectral DAPI (PerkinElmer); neural/glial antigen 2 (NG2) (R&D systems, Minneapolis, USA). For metastases at least nine independent sections of each lung were stained with Mayer's hemalum (Merck, Darmstadt, Germany) and analyzed. Secondary antibody controls for each antibody species were routinely included (Supplementary Figure 1 ).
9
Protein Expression Analysis Protocol
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Cells were washed with cold phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer. Then proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with blocking buffer, followed by primary antibodies against vimentin (1:1000, Abcam), alpha-smooth muscle actin (α-SMA; 1:1000, Sigma, St. Louis, USA), TSG6 (1:100, R&D), I-α-I (1:1000, Dako, Carpinteria, CA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000, CST) as well as horseradish peroxidase (HRP)-labeled secondary antibodies. ECL method was used to detect the specific bands.
10
Glucose Uptake Assay in Smooth Muscle Cells
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Incubated cells on cover glasses were used for each uptake experiment. After incubation, medium was changed to No-glucose D-MEM (Wako) containing 1 mM 2-(N
(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) (Cayman Chemical, Ann Arbor, MI, U.S.A.), hoechst33342 (1:500) (Wako) and 65 mM KCl.
Phloridzin was added at the same time, in concentrations of 10 and 100 µM. After 20 min, buffer was removed and rinsed three times with D-PBS
buffer (Wako). Cells were incubated with 4% paraformaldehyde for 30 min and 2% BSA for 20 min in D-PBS buffer at 4°C. After incubation, cells were incubated
with alpha-smooth muscle actin (α-SMA) (Sigma-Aldrich, St. Louis, MO, U.S.A.) overnight. Incubated cells were rinsed with D-PBS, and the cover glass was put
onto a glass slide for examination using an Axiovert 200 M fluorescence microscope (Carl Zeiss Japan, Tokyo, Japan). The excitation and emission wavelength to
detect 2-NBDG were 488 nm and 560 nm.
2-NBDG, hoechst33342 and α-SMA fluorescence were detected by the Axiovert 200 M. Hoechst and α-SMA double-positive cells were determined in smooth muscle
cells. Assay of 2-NBDG uptake was determined by measuring the fluorescent intensity using image J software.
(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) (Cayman Chemical, Ann Arbor, MI, U.S.A.), hoechst33342 (1:500) (Wako) and 65 mM KCl.
Phloridzin was added at the same time, in concentrations of 10 and 100 µM. After 20 min, buffer was removed and rinsed three times with D-PBS
buffer (Wako). Cells were incubated with 4% paraformaldehyde for 30 min and 2% BSA for 20 min in D-PBS buffer at 4°C. After incubation, cells were incubated
with alpha-smooth muscle actin (α-SMA) (Sigma-Aldrich, St. Louis, MO, U.S.A.) overnight. Incubated cells were rinsed with D-PBS, and the cover glass was put
onto a glass slide for examination using an Axiovert 200 M fluorescence microscope (Carl Zeiss Japan, Tokyo, Japan). The excitation and emission wavelength to
detect 2-NBDG were 488 nm and 560 nm.
2-NBDG, hoechst33342 and α-SMA fluorescence were detected by the Axiovert 200 M. Hoechst and α-SMA double-positive cells were determined in smooth muscle
cells. Assay of 2-NBDG uptake was determined by measuring the fluorescent intensity using image J software.
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