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. 2023 Jul 28;51(17):9166–9182. doi: 10.1093/nar/gkad631

Figure 5.

Figure 5.

Nuclear HDAC6 is displaced from chromatin and degraded via the proteasome in response to DSBs. (A, B) HeLa cells were exposed to an increasing dose of irradiation (IR) and released after 1 h or treated with 10 μM etoposide (VP16) for the indicated time. Then, whole cell lysate (β-actin), nuclear (Lamin-B1), and chromatin (H3) fractions were isolated and analyzed by western blotting. (C) HeLa cells were treated with 10 μM MG132 for 3 h or 50 μM CHQ for 12 h, then exposed to 10 Gy IR and released for 1 h before analysis of the whole cell lysate and nuclear fractions by western blotting. (D–F) HeLa cells were co-transfected with Flag-HDAC6 and HA-ub (D), Flag-HDAC6 and different HA-ubiquitin constructs (WT: wild type ubiquitin, K48R: K48-mutant ubiquitin, K63R: K63-mutant ubiquitin) (E) or Flag-HDAC6 wild-type (WT) or mutant (K116R) (F) for 48 h. Then, the cells were treated with 10 μM MG132 for 2 h, exposed to 10 Gy IR, and released for 1 h. The nuclear fractions were then subjected to immunoprecipitation with an anti-Flag antibody and analyzed by western blotting.