Fig. 2

SUMO1 modification of KHSRP is regulated by hypoxia, hydrogen peroxide and growth factors. a Hypoxia upregulates SUMO1 modification of KHSRP. 293T cells transfected with His-SUMO1 and HA-KHSRP-WT or HA-KHSRP-K87R were cultured in 1% oxygen condition (hypoxia) for 6, 12 h before cells were harvested. Ni²⁺-NTA resin pull down was performed to detect the SUMO1 modification of exogenous KHSRP. b Hydrogen peroxide (H₂O₂) downregulates SUMO1 modification of KHSRP. 293T cells transfected with His-SUMO1 and HA-KHSRP-WT or HA-KHSRP-K87R were treated with 100 μM of H₂O₂ for 1.5, 3 h before cells were harvested. Ni²⁺-NTA resin pull down was performed to detect the SUMO1 modification of KHSRP. c EGF downregulates SUMO1 modification of KHSRP. 36 h after transfection with His-SUMO1 and HA-KHSRP-WT, 293T cells were starved overnight and then stimulated with EGF (50 ng/ml) for 5 min before lysed for Ni²⁺-NTA pull down, and followed by Western blotting with indicated antibodies. d Insulin downregulates SUMO1 modification of KHSRP. 293T cells transfected with His-SUMO1 and Flag-Ubc9 were treated with insulin (1 μM) for 1 h or LY294002 (25 μM) for 16 h before cells were harvested. Ni²⁺-NTA resin pull down was performed to detect the SUMO1 modification of endogenous KHSRP. p-AKT1 (S473) antibody was used to detect the phosphorylation level of AKT1. e Phosphorylation of KHSRP downregulates SUMO1 modifcation of KHSRP. 293T cells transfected with His-SUMO1 and HA-KHSRP-WT, −K87R, −S193A, −K87R-S193A, −S193D, or -K87R-S193D were lysed for the SUMOylation assay with the Ni²⁺-NTA resin pull down. The band intensities were calculated by ImageJ software and the ratios were quantified (a-d)