Figure 2: USP21 deubiquitinates Nanog.

(a) Flag-Nanog and His-ubiquitin were co-expressed with USP21 WT or USP21-C221A in HEK293T cells. After MG132 (10 μM) treatment for 6 h, the ubiquitinated proteins were pulled down under denaturing conditions using Ni-NTA agarose beads and the ubiquitination of Nanog was detected by western blotting using an anti-Flag antibody. (b) His-USP21 protein was purified from E. coli, Flag-Nanog was purified by immunoprecipitated from HEK293T cells were transfected with Flag-Nanog, followed by immunoprecipitation using an anti-Flag antibody and then subjected to in vitro deubiquitination. (c) E14 cells stably expressing control or USP21 shRNA1 and USP21 shRNA3 were treated with MG132 (10 μM) for 6 h. Nanog was immunoprecipitated with an anti-Nanog antibody and the ubiquitination of Nanog was examined by western blotting using an anti-ubiquitin antibody. USP21 was detected by an anti-USP21 antibody from Abgent. (d) Alignment of the potential ubiquitin site in Nanog from different species. (e) WT or lysine-mutated Nanog and His-ubiquitin were co-expressed in HEK293T cells. After treatment with MG132 (10 μM) for 6 h, the ubiquitinated proteins were pulled down under denaturing conditions using Ni-NTA agarose beads and the ubiquitination of Nanog was detected by western blotting using the anti-Nanog antibody. (f) WT or K138/169R Nanog was expressed or co-expressed with Flag-USP21 in HEK293T cells. After treatment with CHX (10 μg ml⁻¹), the protein level of Nanog was analysed by western blotting (left panel), and statistical analysis was presented on the right. Data are means±s.d. (n=3), ***P<0.001 versus WT (Student’s t-test).