Cell Lysis and Protein Extraction

After the indicated incubation times, HLMVECs were washed once with ice-cold phosphate-buffered saline (Gibco Invitrogen), scraped from the bottom of the plate, collected, and lysed with radio-immunoprecipitation buffer (Boston Bioproducts, Ashland, MA) supplemented with protease inhibitor cocktail, 200 mM of phenylmethylsulfonyl fluoride, 1 mM of EDTA, 1 mM of sodium fluoride, and 1 mM of sodium orthovanadate (all from Sigma-Aldrich) as previously described.^{22 (link),23 (link)}

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Piegeler T., Votta-Velis E.G., Bakhshi F.R., Mao M., Carnegie G., Bonini M.G., Schwartz D.E., Borgeat A., Beck-Schimmer B, & Minshall R.D. (2014). Endothelial Barrier Protection by Local Anesthetics: Ropivacaine and Lidocaine Block Tumor Necrosis Factor-α–induced Endothelial Cell Src Activation. Anesthesiology, 120(6), 1414-1428.

Publication 2014

phosphate-buffered saline radio-immunoprecipitation buffer protease inhibitor cocktail phenylmethylsulfonyl fluoride sodium orthovanadate

Buffer Cold Edta Immunoprecipitation Orthovanadate Phenylmethylsulfonyl fluoride Phosphate Protease inhibitor Saline Sodium Sodium fluoride

Corresponding Organization :

Other organizations : University of Illinois Hospital & Health Sciences System, University of Illinois Chicago, University of Zurich

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Variable analysis

independent variables

Incubation times

dependent variables

Protein expression (from cell lysates)

control variables

Phosphate-buffered saline (Gibco Invitrogen)
Radio-immunoprecipitation buffer (Boston Bioproducts, Ashland, MA) supplemented with protease inhibitor cocktail, 200 mM of phenylmethylsulfonyl fluoride, 1 mM of EDTA, 1 mM of sodium fluoride, and 1 mM of sodium orthovanadate (all from Sigma-Aldrich)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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