Extended Data Fig. 6: Mechanical characterization of Atz-AUTAB-mediated PD-L1 degradation.
From: Chemically engineered antibodies for autophagy-based receptor degradation
a-c, Degradation of exogenously expressed PD-L1 assessed by western blotting in HA-PD-L1 stably expressing HeLa cells following treatment as in Fig. 3a–c. d, Western blot of PD-L1 in MDA-MB-231 cells treated with 6.25 nM Atz-AUTAB for 24 h in the presence or absence of 200 μM chloroquine. e, Western blot analysis of PD-L1 and LC3B in MDA-MB-231 cells treated with indicated concentrations of Atz, L25K PEI, and Atz-AUTAB for 12 h and followed by release for 12 h. f, MDA-MB-231 cells with or without siRNA mediated knockdown of LC3B were subjected to treatment with 6.25 nM Atz-AUTAB for 12 h. The PD-L1 levels were detected by immunoblotting. g, Western blot analysis of GABARAP, GABARAPL1, GABARAPL2 in LC3C knockout MDA-MB-231 cells treated with or without 6.25 nM Atz-AUTAB for 12 h. h, Western blot of PD-L1 in MDA-MB-231 cells treated with 6.25 nM Atz-AUTAB for 12 h under siRNA-mediated knockdown of GABARAP, GABARAPL1, and GABARAPL2 respectively. i, HA-PD-L1 stable expressing HeLa cells transfected with scramble or LC3C, GABARAP, GABARAPL1, and GABARAPL2 siRNA were incubated with 100 nM Atz-AUTAB for 1 h. Cells were then fixed and co-stained with antibodies against galectin-3, Lamp1 and Atz. The enlarged images of the white box are presented below, with arrowheads indicating the colocalization of galectin-3, Lamp1 and Atz-AUTAB. The quantification is shown on the right (n = 15 cells per group, means ± s.d.). j, Western blot of PD-L1 in MDA-MB-231 cells treated with 6.25 nM Atz-AUTAB for the indicated time (0, 12, 16, 20 h) under siRNA-mediated knockdown of ATG4B. Scale bars, 10 μm. Statistical significance was calculated via unpaired two-tailed Student’s t-test (i).
