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. 2016 Oct 26;45(3):1144–1158. doi: 10.1093/nar/gkw1025

Figure 2.

Figure 2.

H2Bub1 levels are decreased during starvation-induced autophagy activation mediated by its deubiquitinase USP44. (A) H2Bub1 is decreased in response to starvation as analyzed by western blot assay. 293T cells were starved by HBSS treatment for the indicated periods to activate autophagy. Cell extracts were then prepared and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (B) H2Bub1 is decreased in response to starvation as analyzed by immunofluorescence assay. Immunofluorescence assays were performed using control or starved 293T cells with anti-H2Bub1 antibody. DAPI staining was used to indicate the cell nucleus. (C) USP44 is increased in response to starvation. Control or HBSS-treated (starvation) 293T cells were lysed and subjected to western blot and RT-PCR analyses using specific antibodies or primers, as indicated. The experiments were repeated more than three times (n > 3). (D) USP44 regulates the starvation-induced down-regulation of H2Bub1. 293T cells were transfected with control or USP44-specific shRNAs for 48 h. The cells were then treated with or without HBSS (starvation) for the indicated periods. Cell extracts were prepared and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (E) Loss of H2Bub1 does not depend on autophagy activity. Control and ATG5 RNAi 293T cells were treated with or without starvation for 10 h. The cells were then lysed and subjected to western blot assays using the indicated antibodies. The experiments were repeated more than three times (n > 3). (F) H2Bub1 does not respond to rapamycin treatment. 293T cells were treated with starvation or 2 μM rapamycin for 10 h. The cells were then lysed and subjected to western blot assays using the indicated antibodies. The experiments were repeated more than three times (n > 3). (G) H2Bub1 likely functions upstream of H4K16ac in the regulation of autophagy. Cell extracts from 293T cells transfected with RNF20 siRNA, H2BK120R mutant plasmid, ATG5 siRNA, or hMof siRNA were prepared. Western blot analysis was then performed using the indicated antibodies. The experiments were repeated more than three times (n > 3).