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. 2017 Nov 15;38(4):727–740. doi: 10.1177/0271678X17740031

Figure 1.

Figure 1.

BBB endosome isolation and SRM analysis. (a) Workflow outlining the method for isolation of endosomes for analysis with LC-SRM. Details are described in Methods section. (b) Relative levels clathrin (Cltc), caveolin-1 (Cav1) and flotilin-1 (Flot1) and -2 (Flot2), markers various types of endocytic vesicles (clathrin-coated pits, caveolae and clathrin-independent endocytic vesicles, respectively) in endosomal fractions of BBB SV-ARBEC cells exposed to 5 mg/ml of Bi-FC5Fc for 45 min. The levels are determined using multiplexed LC-SRM. Shown are relative abundance (mean ± SD) of protein-specific peptides values from three endosome preparations. Fractions 1–4 are designated low-density fractions (LDFs); fractions 4–7 high-density fractions (HDFs); fractions 7–10 vHDFs. (c) Levels of clathrin and caveolin-1 in the same endosomal fractions detected by Western blot. (d) Levels of bivalent BBB-crossing antibody Bi-FC5Fc in the same endosomal fractions detected by Western blot.