Figure 4: SRSF3 is a novel target for stress-induced neddylation in vivo.

(a) HEK293T cells were transiently co-transfected with indicated plasmids. After 36 h, cells were treated with either mock or arsenite, lysed under denaturing condition and affinity-purified using Ni²⁺-NTA agarose beads (see Methods). The precipitates were then blotted against anti-Flag antibody. (b) HEK293T cells transfected with empty vector or Flag–SRSF3 were treated with arsenite, lysed under denaturing condition (1% SDS) and immunoprecipitated using Flag beads (see Methods). (c) In vivo neddylation assay was carried out for cells treated with arsenite in a dose-dependent manner (0.15 to 1 mM) for 1 h. (d) In vivo neddylation assay was performed for cells treated with 0.2 mM arsenite at different time points (0–60 min) as indicated. (e) HEK293T cells transfected with indicated plasmids were treated with 0.2 mM arsenite in lane 3 and 4 for 30 min and 60 min, respectively. Cells in lanes 5 and 6 were treated with 0.2 mM arsenite for 60 min and then allowed to recover from stress by changing with fresh medium for 90 min (lane 5) and 180 min (lane 6) and subjected to neddylation assay. (f) NAE inhibitor MLN4924 significantly attenuates SRSF3 neddylation. HEK293T cells pretreated with DMSO, MLN4924 (1 μM) for 18 h or MLN4924 (3 μM) for 1 h prior to arsenite treatment were subjected to neddylation assay. (g) SiRNA mediated knockdown of UBE2M reduces SRSF3 neddylation. In vivo neddylation assay was performed for HEK293T cells transfected with siCONT or siUBE2M under arsenite stress. (h) Ectopic expression of UBE2M-C111S displays dominant negative effect on SRSF3 neddylation. HEK293T cells co-transfected with indicated plasmids were treated with arsenite and subjected to neddylation assay. (i) UBE2M interacts with SRSF3 under arsenite stress. Empty vector or Flag–SRSF3 transfected cells were treated with arsenite, lysed in IP buffer and immunoprecipitated using Flag agarose beads. The precipitates were subjected to western analysis using anti-UBE2M antibody. (j) NEDP1 overexpression inhibits SRSF3 neddylation. HEK293T cells transfected with indicated plasmids were treated with arsenite and affinity purified using Ni²⁺-NTA agarose beads.