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. 2021 Sep 27;11:711735. doi: 10.3389/fonc.2021.711735

Figure 5.

Figure 5

Otubain 2 (OTUB2) increases the stability of KDM1A. (A) Relative protein levels of KDM1A in GC cells transfected with OTUB2 overexpression plasmids (MKN-45 and SGC-7901 cells) or shRNA targeting OTUB2 (HGC-27 and AGS cells). Actin served as internal control. (B) co-immunoprecipitation was conducted with anti-Flag (Flag-OTUB2) or anti-HA (HA-KDM1A) antibody and was analyzed by western blotting in 293T cells. (C) after transfected with shOTUB2, cells were treated with or without MG132. The ubiquitination level of KDM1A in MKN-45 and HGC-27 cells was measured by Western blot. Actin served as internal control. (D) After transfected with OTUB2 overexpression plasmids (MKN-45 cells) or shOTUB2, cells were treated with or without cycloheximide (CHX) for 0, 1, 2, 4, and 8 h, respectively. The protein expression levels of KDM1A were assessed by Western blot. Actin was used as internal control. Data was represented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.