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In the latter case, proteins are then routed to the plasma membrane through a different set of carriers, including recycling endosomes (Spang, 2015).
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In either case, proteins were separated under nonreducing conditions.
The clustering of major protein groups is indicated by the color code used in Figure 2. In many cases proteins are either down- (green) or up-regulated (red).
In those cases, proteins were listed according to the number of identified peptides.
In certain cases, proteins were found to be decreased in both CFS and nPTLS compared to healthy normal controls.
In both cases, proteins were extracted using NP40 supplemented with X100 protease and phosphotase inhibitors (Calbiochem and Sigma respectively) and abundance was measured using a Bradford assay (Sigma).
In some cases, proteins were treated with endoglycosidase H or PNGase F (New England Biolabs).
In all cases, protein was localized at both the plasma membrane and in intracellular vesicular-like structures (data not shown) suggesting that mutation of these cytoplasmic tyrosine residues to phenylalanine did not grossly perturb FLRT1 expression or intracellular trafficking.
In some cases, protein was purified without spin-labeling for functional work.
In some cases, protein was isolated 48h after transfection and the level of silencing determined by Western Blot.
In all cases protein was incubated with lipids for up to 30 min and fluorescence measured with excitation of 295 nm (slit 5 nm).
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