Fig. 1

KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni²⁺-NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli-based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli BL21 (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni²⁺-NTA resin