Extended Data Fig. 8: Synthesis of anti PD-L1 glycopolypeptide conjugates, PD-L1 degradation, and CD71 degradation depends on M6P binding. | Nature

Extended Data Fig. 8: Synthesis of anti PD-L1 glycopolypeptide conjugates, PD-L1 degradation, and CD71 degradation depends on M6P binding.

From: Lysosome-targeting chimaeras for degradation of extracellular proteins

Extended Data Fig. 8

a, Anti-PD-L1 was non-specifically labelled with BCN, then incubated with poly(M6Pn)short for 3 days at room temperature. Reaction progress was monitored by native gel electrophoresis and visualized by Coomassie stain. b, Cell-surface PD-L1 determined by live-cell flow cytometry after incubation with anti-PD-L1 or conjugates (50 nM). At each time point, cells were washed, lifted, brought to 4 °C, then stained for PD-L1 using excess unconjugated anti-PD-L1 (1 μM). c, PD-L1 levels in MDA-MB-231 cells after 48-h incubation with anti-PD-L1 or Ab-3. d, PD-L1 levels in HDLM-2 cells after 36 h incubation with anti-PD-L1 or Ab-3. e, Quantification of PD-L1 degradation in HDLM-2 cells with Ab-3f, Atezolizumab was non-specifically labelled with NHS-(PEG)4-N3, then incubated with poly(M6Pn)short-BCN for 3 days at room temperature. Reaction progress was monitored by native gel electrophoresis and visualized by Coomassie stain. g, Levels of CD71 in Jurkat cells after 24 h in the presence of 5 mM M6P. Data are representative of two (a, c, f, g) independent experiments. For b, data are mean ± s.d. of three independent experiments, and cell surface levels are relative to untreated cells. For e, data are mean ± s.d. of three independent experiments, one of which is shown in d. Per cent control was calculated by densitometry and normalized to loading control.

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