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Notably, the percentage of overlap in hydrophobic proteins with a GRAVY score above 0 was 78%, suggesting that crude membrane fractionation is suitable for enriching membrane proteins with high hydrophobicity.
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Indicated membrane fractions obtained from the membrane fractionation were incubated with cytosol for the LC3 lipidation assay.
Plasma membrane fractionation was performed using a membrane protein extraction kit from BioVision.
Membrane fractionation was performed as previously described.
Membrane fractionation was as described (Garnett et al., 2005).
Membrane fractionation was performed as previously described (Smith et al, 2005).
Membrane fractionation was performed using an OptiPrep™ gradient (AXIS-Shield, Oslo, Norway).
Procedures for membrane fractionation were adapted from the method of Macdonald and Pike.
The procedure for membrane fractionation was essentially as described for the Nycodenz fractionation, except for the homogenization buffer used (25 mM Tris pH6.8, 150 mM NaCl, 1 mM PMSF, 20 μg/μl of leupeptin and 1 μg/μl of aprotinin).
For further outer-membrane fractionation, the pellet (total membrane fraction) was resuspended sterilized distilled water.
Expression of NADPH oxidase (NOX) subunit gp91 phox in membrane fractions was assessed with subcellular fractionation.
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