Fig. 1. STK38 recognizes monoufmylated H4.

(A) UFM1 binding motif in STK38 protein. (B) STK38 interacts with the SUV39H1–KAP-1–HP1 complex. Rabbit immunoglobulin G (IgG) or STK38 antibody immunoprecipitates from U2OS cells were blotted with indicated antibodies. (C and D) Purified GST or GST-STK38 proteins were incubated with irradiated U2OS cell lysates for 1 hour and then blotted with indicated antibodies. (E) Purified UBA5, UFC1, UFL1, UFM1, and H4 proteins were incubated together in the presence of ATP and MgCl₂ at 30°C for 90 min. Reaction products were used for GST pull-down assay with purified wild-type (WT) or mutant GST-STK38 proteins. (F) U2OS cells expressing WT or mutant STK38 were lysed, treated with Benzonase, and then subject to immunoprecipitation with HA agarose beads. Immunoprecipitates were blotted with indicated antibodies. (G and H) The mononucleosomes were purified from WT and K31R H4–expressing U2OS cells with/without 10 Gy IR (G) or U2OS cells transfected with control siRNA, STK38 siRNA, or UFM1 siRNA with/without 10 Gy IR (H) and then incubated with STK38 antibody. The immunoprecipitates were blotted with indicated antibodies. *Maxi PAGE gels that were used to separate the endogenous and exogenous H4. Mini PAGE gels were used for the rest of the blots.