Fig. 5

ANXA1 regulates glutamine metabolism via deubiquitination and protein stability of GOT1. A: RT-PCR detecting the mRNA levels of GOT1 after knockdown of ANXA1 in HUCCT1 and HCCC9810 cells. B: Western blot detecting the expression of GOT1 in HUCCT1 and HCCC9810 cells with ANXA1 knockdown. C: IB of GOT1, ANXA1, and α-tubulin in HUCCT1 cells transduced with sh-ANXA1#1 or sh-control after CHX treatment (100 µg/ml) for the indicated times (left). A graph showing normalized GOT1 levels is also shown (right). D-E: IB of GOT1, ANXA1, and α-tubulin in HUCCT1 cells transduced with sh-control or sh-ANXA1 after treatment with MG132 (10 μm, top) or CQ (50 μm, bottom). F: IP (using anti-Flag antibody) and IB of HA-Ub, Flag, ANXA1, α-Tubulin, and GOT1 in RBE cells transfected with the indicated plasmids.(k48O refers to ubiquitin expression vector mutant plasmid that retains only the K48 site, k63O refers to ubiquitin expression vector mutant plasmid that retains only the K63 site). G: IP (using anti-Flag antibody) and IB of HA-Ub(k48O), Flag, ANXA1, α-Tubulin, and GOT1 in HUCCT1 and HCCC9810 cells transfected with the indicated plasmids after treatment with MG132 (10 μm, 6 h). H: Glutamine uptake and glutamate/ aspartate levels were examined in HUCCT1 and HCCC9810 cells with stable of knockdown ANXA1 or overexpress GOT1. I: Mechanism diagram of GOT1-mediated glutamine metabolism. J: GSH and ROS levels were examined in HUCCT1 and HCCC9810 cells with stable of knockdown ANXA1 or overexpress GOT1. Data are representative of three (A, C(right), H, J) independent experiments. One-way ANOVA (A, H, J); Two-way ANOVA (C(right)). **, p < 0.01; ***, p < 0.001; ns, not significant. Data are mean ± SD