HEK 293 cells were purchased from ATCC while the stably transfected HEK 293-hUT cell line was generated in our laboratory (23 ). Cell culture reagents were obtained from Invitrogen while antibodies used to evaluate ERK1/2 phosphorylation, i.e., a rabbit polyclonal antibody against phospho-p44/42 MAPK and an anti-total MAP kinase antibody, were purchased from Cell Signaling Technology. α-Actin antibody was obtained from Abcam. The IP-One ELISA assay kit from CisBio Bioassays. The fluorenylmethyloxycarbamate- (Fmoc-) protected amino-acids, Rink amide AM resin (with Nle), N,N′-diisopropylcarbodiimide (DIC), and ethyl cyanohydroxyiminoacetate (Oxyma) were purchased from Chem-Impex. Trifluoroacetic acid (TFA), methanol (MeOH), acetonitrile (ACN), diethyl ether, N,N-dimethylformamide (DMF), piperidine, dichloromethane (DCM), and cell mask were obtained from Fisher Scientific. YM254890 was purchased from Cedarlane. All other chemicals, including 99.9% 2H2O, were from Sigma-Aldrich. 98% DPC-d38 was obtained from Cambridge Isotope Laboratories, Inc, and [(2,2,3,3-tetradeuterio)-3-(trimethylsilanyl)]propionic acid (TSP) from MSD Isotopes.
α actin antibody
Manufactured by Abcam
Sourced in United States
The α-actin antibody is a protein-specific antibody that recognizes the alpha-actin protein. Alpha-actin is a structural protein found in the cytoplasm of eukaryotic cells and plays a key role in the maintenance of cell shape and motility.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview 1000 confocal microscope, by Olympus (2 mentions)
Alexa fluor 594 goat anti mouse antibody, by Abcam (2 mentions)
Cellmask, by Thermo Fisher Scientific (1 mentions)
Ip one elisa assay kit, by PerkinElmer (1 mentions)
N n diisopropylcarbodiimide dic, by Chem-Impex (1 mentions)
Dpc d38, by Cambridge Isotopes (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Dichloromethane dcm, by Thermo Fisher Scientific (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Stripping buffer, by Thermo Fisher Scientific (1 mentions)
Iblot dry transfer system, by Thermo Fisher Scientific (1 mentions)
Sirna, by Horizon Discovery (1 mentions)
Alexa fluor 594 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Biotinylated secondary antibody, by Abcam (1 mentions)
A confocal microscope, by Zeiss (1 mentions)
α sarcomeric actin, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
6 protocols using α actin antibody
1
HEK 293 Cell Protein Phosphorylation
2
Syndapin2 Overexpression and Knockdown
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PC12 cells were transfected with Syndapin2-GFP, or siRNA (Dharmacon) using Lipofectamine 2000, and protein was isolated ∼24 h later. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (1% Anatrace, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA and 1 mM phenylmethylsulfonyl fluoride [PMSF] in PBS) on ice for 30 min. Lysates were centrifuged at 12,000 × g at 4°C for 10 min. Supernatants were boiled in lithium dodecyl sulfate (LDS) sample buffer containing 62.5 mM dithiothreitol (DTT) for 10 min and loaded onto 4–12% Tris-Bis gels (NuPAGE). Protein was transferred onto nitrocellulose membrane using the iBlot dry transfer system (ThermoFisher Scientific), and syndapin2 detected with monoclonal antibody (ThermoFisher Scientific) and peroxidase-labeled secondary antibody. Blots were stripped with stripping buffer (ThermoFisher Scientific) and reprobed with α-actin antibody (Abcam).
3
Histological Analysis of Murine Hearts
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Hearts from mice at 1 week and 8 weeks after TAC were fixed with formalin, embedded with paraffin and cut into 4 μm slices for histological analysis. The size of heart was assessed by histological analysis with hematoxylin / eosin (HE) staining and Masson staining.
Paraffin-embedded tissue sections were stained with mouse monoclonal α-sarcomeric actin (α-actin) antibody (1:75, Abcam) and Alexa Fluor 594 goat anti-mouse antibody (1:200, Molecular Probes) as secondary antibody. DAPI (4’-6-diamidino- 2-phenylindole, Sigma) were used as nuclei marker. Sections were mounted and analyzed with a fluoview 1000 confocal microscope (Olympus, Japan).
Paraffin-embedded tissue sections were stained with mouse monoclonal α-sarcomeric actin (α-actin) antibody (1:75, Abcam) and Alexa Fluor 594 goat anti-mouse antibody (1:200, Molecular Probes) as secondary antibody. DAPI (4’-6-diamidino- 2-phenylindole, Sigma) were used as nuclei marker. Sections were mounted and analyzed with a fluoview 1000 confocal microscope (Olympus, Japan).
4
Myocardial Tissue Immunofluorescence Analysis
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First, the myocardial tissue samples were fixed by immersion in 4% paraformaldehyde for 24 to 48 h. Then, myocardial tissue was embedded in paraffin and stained with haematoxylin and eosin (H&E). The cardiomyocyte and paraffin-embedded tissue sections were incubated with a mouse monoclonal (α-actin) antibody (1:100, Abcam, LA, USA) for at 4°C overnight. An Alexa Fluor 594 goat anti-mouse antibody (1:200, Abcam, LA, USA) was used as a secondary antibody. DNA in the nucleus of cardiomyocytes was stained with 0.025 μg/ml DAPI at room temperature without any exposure to light. Finally, the slides were mounted with Vectashield and 4',6-diamidino-2-phenylindole mounting medium without being exposed to light at 4°C overnight, and fluorescently labelled cells were closely scrutinized using a fluoview 1000 confocal microscope (Olympus, Osaka, Japan). A random collection of 10 cardiomyocytes images was created for the calculation of cardiomyocyte area using Image J software. Quantification of fluorescence intensities was carried out using MetaMorph software (Boyce Scientific, St. Louis, MO).
5
Immunocytochemical Analysis of α-Actin
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The cell density was adjusted to 2×104 cells/mL, and the cells were cultured on a cover glass. Then, the cover glass was fixed with 4% paraformaldehyde, cleared with PBS containing 0.5% Triton X-100, treated with 3% hydrogen peroxide (H2O2) in methanol to quench endogenous peroxidase activity, and blocked in 6% goat serum. The samples were incubated overnight with an α-actin antibody (Abcam, USA; diluted1:200) at 4°C. Then, the samples were incubated with a biotinylated secondary antibody (Abcam, USA; diluted 1:200) at 37°C for 30 min. HRP-labeled streptavidin and the HRP substrate were added, and the samples were incubated at 37°C for 30 min. A 3,3’-diaminobenzidine (DAB) (ZSGB-BIO, China) substrate system was used for color development in the dark. After counterstaining with hematoxylin, the sections were dehydrated for 5 min, dehydrated with an alcohol gradient, cleared with xylene, and mounted with neutral mounting medium.
6
Histological Analysis of Mouse Heart Post-TAC
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Mouse heart tissues were collected 4 weeks after TAC. The heart was fixed with paraformaldehyde, embedded in paraffin and cut into slices of 4 μm thickness for histological analysis. The size and morphological alterations of the heart tissue were assessed by hematoxylin/eosin (HE) and Masson trichrome staining. Paraffin-embedded tissue sections were stained with mouse monoclonal α-sarcomeric actin (α-actin) antibody (1:75, Abcam) and Alexa Fluor 594 goat anti-mouse antibody (1:200, Abcam) as secondary antibody. Cell nucleus was stained with DAPI (Sigma-Aldrich, Merck KGaA). Sections were mounted and analyzed under a confocal microscope (Zeiss, GmbH). The heart tissues collected from six mice in each group were analyzed [20 (link)].
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