Fig. 3: K26 SUMOylation-defective mutation of YBX1 significantly enhanced YBX1 binding with DDX5 protein.

A Effects of YBX1 and its mutants on AKT signaling in DLD-1 cells transfected with indicated plasmids. B YBX1-binding proteins were enriched from total lysates of HEK293T cells with over-expression of Flag-YBX1 with anti-Flag magnetic beads, separated by SDS-PAGE and stained by Coomassie brilliant blue. The specific binding protein bands (shown as arrows) were subjected to MS/MS analysis. C The MS/MS identification parameters of YBX1 and its eight candidate binding partners, including IGF2BP1, HNRPU, IGF2BP3, DDX5, G3BP1, YTHDF3, IGF2BP2 and ENOA proteins. D Interaction of YBX1 with its eight candidate binding partners (GF2BP1, HNRPU, IGF2BP3, DDX5, G3BP1, YTHDF3, IGF2BP2 and ENOA) was validated in HEK293T cells transfected with indicated plasmids. E Interaction of YBX1^K26R or YBX1^4KR with IGF2BP1, HNRPU, IGF2BP3, DDX5, G3BP1, YTHDF3, IGF2BP2 was compared to that of the wild-type YBX1 in HEK293T cells transfected with indicated plasmids. F The interaction between DDX5 with YBX1, YBX1^K26R, YBX1^4KR was measured in CRC cells transfected with indicated plasmids. G Flag-YBX1 or Flag-YBX1^K26R overexpression had influence on p-AKT level in CRC cells stably transfecting with sh-DDX5. IP immunoprecipitation, Input Same account of cell lysate to load, sh-Control short hairpin RNA targeting to empty vector, sh-DDX5 short hairpin RNA targeting to DDX5.