Cells were trypsinized and washed in cold PBS before being lysed in 2X Laemmli sample buffer containing 0.5% beta-mercaptoethanol, 1% each of Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 2, and Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich, P8340, P5726, P0044, respectively). Samples were run on 4-20% Tris-Glycine gels (Life Technologies) and transferred to 0.45 μm PVDF-FL membrane (Millipore). Membranes were blocked in 2% milk solution and probed with the following antibodies: NMIIB (Biolegend, rabbit pAb; 1:1000 cat #909901), GAPDH (Santa Cruz Biotechnology rabbit pAb; 1:5000). EGFR (Cell Signaling Technology, rabbit pAb; 1:1000). Blots were analyzed with LiCor Odyssey CL imaging system.
Nmiib
Manufactured by BioLegend
NMIIB is a non-muscle myosin-II protein. It is a member of the myosin II family, which are responsible for various cellular processes such as cell division, cell migration, and muscle contraction.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using nmiib
1
Immunoblotting of Cell Signaling Proteins
2
Immunolabeling of Cytoskeletal Elements
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Immunolabeling of cytoskeletal elements was performed in isolated basilar papillae, fixed in 4% paraformaldehyde for 30 min at room temperature, blocked in 10% goat serum for 1 h, and labeled with primary antibodies to α2-spectrin (monoclonal; Thermo Fisher, Waltham, MA), β2-spectrin (monoclonal; Sigma-Aldrich), and the nonmuscle myosin NMIIB (MYH10 gene, polyclonal; Biolegend) all at 1:100 dilution for 2 h at room temperature. Preparations were then labeled with secondary antibodies (rabbit or mouse conjugated with Alexa-488 or Alexa-568, 1:300, 1.5 h) and with Alexa-647 phalloidin, mounted using Fluoromount G and viewed on a Nikon A1 confocal microscope with ×100, 1.4 numerical aperture objective.
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