OTUB2 Antibody - #AF9147

Figure 2 Otubain 2 (OTUB2) promotes the maintenance of gastric cancer (GC) stem cell properties. (A) Relative protein expression levels of OTUB2 in GC cells (MKN-45, SGC-7901, SNU-1, HGC-27, and AGS) and gastric epithelial cells-1 (GES-1) were detected by Western blot. Actin served as internal control. (B) The protein levels of OTUB2, Nanog, ALDH, and Oct4 in GC cells were measured by Western blot after transfected with OTUB2 overexpression vector (MKN-45 and SGC-7901 cells) or shRNA targeting OTUB2 (HGC-27 and AGS cells). Actin served as internal control. (C) The effects of OTUB2 overexpression (MKN-45 and SGC-7901 cells) or knockdown (HGC-27 and AGS cells) on spheroid-forming ability were detected by sphere formation assay. (D) After cells were transfected with OTUB2 overexpression vector (MKN-45 and SGC-7901 cells) or shRNA targeting OTUB2 (HGC-27 and AGS cells), percentage of CD44 and CD133 double positive cells was detected by flow cytometry. Data was represented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3 Otubain 2 (OTUB2) promotes the proliferation, invasion, and migration of gastric cancer (GC) cells. (A) Cell viability was detected in OTUB2-overexpressed (MKN-45 and SGC-7901 cells) or downregulated GC cells (HGC-27 and AGS cells). (B) Proliferating OTUB2-overexpressed (MKN-45 and SGC-7901 cells) or downregulated GC cells (HGC-27 and AGS cells) were labeled with EdU (red); cell nuclei were stained with DAPI (blue). Wound healing assays (C) and Transwell assays (D) were used to determine the changes in migratory and invasive abilities of GC cells transfected with OTUB2 overexpression vector (MKN-45 and SGC-7901 cells) or shRNA targeting OTUB2 (HGC-27 and AGS cells). Data was represented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4 Otubain 2 (OTUB2) promotes xenograft tumor growth in vivo. (A) The nude mice were injected with GC cells, which were transfected with OTUB2 overexpression vector (SGC-7901 cells) or sh-OTUB2 (HGC-27 cells), respectively. Tumor volumes were calculated after injection every 5 days, tumor weight was measured 35 days after injection. (B) The tumor sections were underimmumohistochemical staining using antibodies against OTUB2 or Ki-67. Data was represented as mean ± SD. **P < 0.01.

Figure 5. OTUB2 deubiquitinated TRAF6 and activated AKT. (A) Bioinformatics analysis of interaction molecules of OTUB2; (B) Co-IP with anti-Flag antibody showing interactions between exogenous OTUB2 and TRAF6 in HEK293T cells; (C) qPCR analysis was performed to assess the mRNA levels of TRAF6 in MDA-MB-231 and BT-549 cells following OTUB2 overexpression; (D) OTUB2 promoted TRAF6 protein expression in a dose-dependent manner; (E) HEK293T cells transfected with Flag-TRAF6, HA-Ub and Myc-OTUB2 or the empty plasmids following MG132 treatment (10 μm, 6 h) were subjected to denatured-IP and immunoblotted with the indicated antibodies; (F) UbiBrowser 2.0 was used to predict substrates of E3 ligase; (G) Western blot analysis was performed to assess AKT, p-AKT, and TRAF6 protein expression levels in OTUB2 knockdown MDA-MB-231 and BT-549 cells (ns: p > 0.05).