Extended Data Fig. 2: Proteomics and subcellular fractionation.
From: Interactions between TTYH2 and APOE facilitate endosomal lipid transfer
a, Scheme of the pull-down experiment from HEK293 extracts with Sb1 immobilized on streptactin resin. The eluted sample was analyzed by LC/MS for presence of TTYH2 and its interaction partners. b, Table summarizing the results of the pulldown LC/MS analysis with TTYH2 and APOE identified as major co-purifying components. A sybody binding LRRC8A62 was used in a negative control experiment where neither APOE nor TTYH2 were detected. c, Subcellular fractionation of the post nuclear supernatant of mouse neuroblastoma cells (N2A) on a sucrose gradient. TTYH2 (red) and specific marker proteins defining the cellular origin of the fractions (illustrated by shaded background) were identified by western blot. Plotted are averaged intensities from n = 3 independent fractionation experiments. that are normalized to the highest value. Errors are s.e.m. d, e, Sections of representative western blots from a fractionation of post-nuclear supernatant from HEK293 (d) or N2A cells (e) on a sucrose gradient. TTYH2 and APOE are co-localizing in the same fractions overlapping with endosomal markers in HEK293 cells (d). A similar colocalization of TTYH2 with endosomal markers is found in N2A cells (e). f, western blots showing recognition of purified TTYH2 by two anti-TTYH2 antibodies used in western blots (Ab1) and immunocytochemistry (Ab2). Each experiment was performed once. g, Western blot showing recognition of purified APOE by an antibody used in western blots and immunocytochemistry, the experiment was carried out once. h-i Uncropped western blots of APOE (h) and TTYH2 (i) in subcellular fractions of HEK293 cells shown in d. j, Uncropped Western blot of TTYH2 in subcellular fractions of N2A cells shown in e. f-j, TTYH2 location is indicated by ‘*’, APOE location by ‘#’. The western blots shown in h-j were repeated 3 times in separate fractionation experiments. The diagram in a was created using BioRender (https://www.biorender.com).
