Skip to main content
. Author manuscript; available in PMC: 2018 May 3.
Published in final edited form as: Cell Rep. 2018 Apr 17;23(3):823–837. doi: 10.1016/j.celrep.2018.03.078

Figure 2. ZRANB1 Is an EZH2 Deubiquitinase.

Figure 2

(A) HEK293T cells were co-transfected with MYC-EZH2, MYC-GFP, and SFB-ZRANB1; treated with 50 μg mL−1 cycloheximide (CHX); harvested at different time points; and then immunoblotted with antibodies against MYC and FLAG. MYC-GFP serves as the control for transfection.

(B) LM2 cells were transfected with ZRANB1 siRNA, treated with 50 μg mL−1 CHX, harvested at different time points, and then immunoblotted with antibodies against EZH2 and β-actin.

(C) ZRANB1-knockout HEK293A cells were co-transfected with MYC-EZH2 and SFB-ZRANB1, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against ubiquitin (Ub) and MYC. Cells were treated with 10 μM MG132 for 6 hr before collection. Before immunoprecipitation, lysates were heated at 95°C for 5 min in the presence of 1% SDS (for denaturing), followed by 10-fold dilution with lysis buffer and sonication.

(D) Ubiquitinated MYC-EZH2 was purified with anti-MYC beads from ZRANB1-knockout HEK293A cells co-transfected with MYC-EZH2 and HA-ubiquitin (Ub), and it was incubated with His-ZRANB1 purified from insect cells. After the in vitro deubiquitination reaction, the bound proteins were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against HA and EZH2.

(E) Control and ZRANB1-knockout HEK293A cells were co-transfected with MYC-EZH2 and lysine-specific mutants of HA-ubiquitin (Ub), followed by immunoprecipitation with anti-HA beads and immunoblotting with antibodies against EZH2 and HA. Cells were treated with 10 μM MG132 for 6 hr before collection. Before immunoprecipitation, lysates were heated at 95°C for 5 min in the presence of 1% SDS (for denaturing), followed by 10-fold dilution with lysis buffer and sonication.

(F) K33 linkage-specific ubiquitinated MYC-EZH2 was purified with anti-MYC beads from ZRANB1-knockout HEK293A cells co-transfected with MYC-EZH2 and the K33-specific mutant of HA-ubiquitin (Ub), and it was incubated with His-ZRANB1 purified from insect cells. After the in vitro deubiquitination reaction, the bound proteins were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against HA and EZH2.

See also Figure S3.