Extended Data Fig. 4: Ab-1 mediates uptake of soluble proteins to lysosomes. | Nature

Extended Data Fig. 4: Ab-1 mediates uptake of soluble proteins to lysosomes.

From: Lysosome-targeting chimaeras for degradation of extracellular proteins

Extended Data Fig. 4

a, Uptake of an Alexa Fluor-488 (AF488)-labelled mouse IgG (m-IgG-488) into cells using antibody LYTACs. b, Uptake of m-IgG-488 using Ab-1. Mean fluorescence intensity (MFI) fold change over background uptake measured by live-cell flow cytometry. K562 cells were incubated at 37 °C for 1 h with 50 nM IgG-488 or 50 nM IgG and 25 nM of anti-mouse or Ab-1. c, AF488 signal (green) colocalized with acidic endosomes and lysosomes as labelled with LysoTracker Red (magenta). Expanded view shows a cell containing IgG-488 and LysoTracker Red. Scale bar, 20 μm. d, ApoE4-647 uptake over time. K562 cells were incubated with 50 nM ApoE4-647 in the presence or absence of 25 nM anti-ApoE4 and Ab-1. At the indicated time point, cells were aliquoted and median fold intensity (MFI) measurements were measured by live-cell flow cytometry. e, Total protein levels for leupeptin inhibition of apoE4 degradation in K562 cells, corresponding to lanes shown in Fig. 3h. Total protein was visualized by Coomassie stain. f, Flow cytometry plots of ApoE4-647 uptake over time, with or without leupeptin inhibition. g, Uptake of ApoE4-647 to lysosomes. K562 cells were incubated with PBS, 50 nM ApoE4-647, 25 nM anti-ApoE4 and 25 nM Ab-1 for 1 h or 24 h in complete growth media at 37 °C. Alexa Fluor-647 signal (red) colocalizes with acidic endosomes and lysosomes as labelled with LysoTracker Green (turquoise). Data are representative of two (c, eg) independent experiments. Data are mean ± s.d. of three independent experiments (b, d). P values were determined by unpaired two-tailed t-tests; fold changes are reported relative to incubation with protein targets alone (b) or background fluorescence (d).

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