Fig. 2 Contribution of SQSTM1 to the autophagic degradation of ARNTL.

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Fig. 2 Contribution of SQSTM1 to the autophagic degradation of ARNTL. Contribution of SQSTM1 to the autophagic degradation of ARNTL. (A) Western blot analysis of the indicated proteins in HT1080 and Calu-1 cells following treatment with RSL3 (0.5 μM) in the absence or presence of MG-132 (2.5 μM), spautin-1 (5 μM), or chloroquine (50 μM) for 12 hours. (B) Western blot analysis of the indicated proteins in THP1 cells after treatment with TNF (10 ng/ml) in the absence or presence of MG-132 (2.5 μM) for 1 hour. (C) Western blot analysis of the indicated proteins in wild-type, atg5−/−, atg7−/−, and atg9a−/− mouse embryonic fibroblasts (MEFs) after treatment with RSL3 (0.5 μM) for 12 hours. (D) Western blot analysis of the indicated protein expression in control, ATG5 knockdown (ATG5KD), and ATG7 knockdown (ATG7KD) HT1080 cells following treatment with RSL3 (0.5 μM) for 12 hours. (E) Mass spectrometry analysis identified SQSTM1 as a direct binding protein of ARNTL in HT1080 cells. These are gels of the proteins that bind to ARNTL, stained with Coomassie Brilliant Blue. Ab, antibody; IgG, immunoglobulin G. (F) Immunoprecipitation (IP) analysis of ARNTL-binding proteins in HT1080 cells following treatment with erastin (10 μM) or RSL3 (0.5 μM) for 6 hours. IB, immunoblot. (G) Western blot analysis of the indicated proteins in wild-type cells, sqstm1−/− MEFs, or sqstm1−/− cells transfected with Sqstm1 complementary DNA (cDNA) (sqstm1−/− + cDNA) following treatment with RSL3 (0.5 μM) for 12 hours. (H) Western blot analysis of the indicated proteins in control and the indicated gene knockdown HT1080 cells following treatment with RSL3 (0.5 μM) for 12 hours. ACTB, actin beta. Minghua Yang et al. Sci Adv 2019;5:eaaw2238 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).