Plasma membranes were prepared as published (51). HLFs (19Lu) or MLFs were treated ± 100 multiplicity of infection (MOI) lentivirus as below, ± RN-1734 (50 μM, 1 hour), and ± TGF-β (2 ng/ml, 3 to 24 hours) and lysed with osmotic lysis buffer [25 mM tris-HCl (pH 7.4), 5 mM EDTA, and protease and phosphatase inhibitors]. Cells were homogenized, toggled at 4°C for 20 min, and centrifuged at 1000g at 4°C for 5 min to remove cell debris/nuclei. Then, the supernatant was centrifuged at 37,000g at 4°C for 30 min to pellet the membrane, which was resuspended in Triton lysis buffer [0.8% Triton X-100, 20 mM tris-HCl (pH 7.4), 300 mM NaCl, 20% glycerol, and protease and phosphatase inhibitors]. The supernatant contains the cytosolic fraction, which includes all intracellular membranes/organelles/cytoskeletal proteins, whereas the pellet contains plasma membrane–associated proteins (18). HLF (19Lu) plasma membranes were further subjected to immunoprecipitation and lipid kinase assays as below, whereas MLF plasma membranes were subjected to Western blotting. Alternatively, a cell surface protein isolation kit used for the biotinylation/isolation of cell surface proteins was purchased and used according to the manufacturer’s instructions (Thermo Fisher Scientific).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.