Fig. 3.

EBP1 controls Suv39H1 stability via UPS. (A) HEK293T cells were transfected with GST-Suv39H1 and Flag-EBP1. Cells were treated with 200 μg/mL cycloheximide (CHX) at indicated time points. Suv39H1 protein level at each point was normalized to Hsp70 antibody abundance. (B) GFP-Suv39H1 and Flag-EBP1 were transfected into HEK293T cells, and cells were then treated with 10 μM DMSO and MG132. After 8 h treatment, cells were lysed and immunoblotted. (C) PC12 cells were transfected with GST-Suv39H1, Flag-EBP1, and HA-Ubiquitin (HA-Ub), and then treated with 10 μM MG132 for 8 h followed by GST pull-down assay. Ubiquitinated Suv39H1 was detected by an anti-HA antibody. (D) PC12 cells were transfected with GST-Suv39H1 and si-Ebp1. After 48 h transfection, cell lysates were subjected to GST pull-down assay. (E) HEK293T cells were transfected by the indicated plasmids. GST pull-down assay was performed. (F) GST-Suv39H1 and Flag-EBP1 were transfected to HEK293T cells, followed by treatment of 10 μM DMSO and MG132 for 8 h. Cell lysates were conducted to GST pull-down assay. **P < 0.01, ***P < 0.001, ****P < 0.0001. Images shown here are representative at least 3 independent experiments and values in these figures represent means ± SEM from more than 3 indepen-dent experiments.