Fig. 3: Neddylation stabilizes SREBP1c protein by competitively interfering with ubiquitination.

a HEK293 cells, which had been cotransfected with Flag-SREBP1c and His-NEDD8 for 48 h, were treated with cycloheximide for the indicated time, and then cell lysates were subjected to Western blotting. Band intensities (mean ± SD, n = 3) on blots were analyzed using ImageJ and plotted. *P < 0.05 versus control group. b HepG2 cells, which were transfected with NEDD8, were treated with OA. After incubated for 24 h, cells were treated with cycloheximide for the indicated time and then subjected to Western blotting. Protein band intensity was analyzed using the ImageJ software. * denotes P < 0.05 versus the control. c HEK293 cells were cotransfected with Flag-SREBP1c, His-NEDD8 and HA-Ubiquitin for 24 h and treated with MG132 (10 μM) for 8 h. After the treatment, immunoprecipitation was performed using anti-Flag beads and assessed by Western blotting. d HEK293 cells were cotransfected with indicated plasmids for 24 h. After transfection, 10 μM of MG132 was added to the cells. Cells lysates were purified with anti-Flag beads, and proteins were analyzed by Western blotting. e The SREBP1c domains and His-NEDD8 plasmids were cotransfected into HEK293 cells. Transfected cells were subjected to Ni²⁺ pull-down assay, and immunoblotted using anti-Flag antibody. f HEK293 cells were cotransfected with the indicated plasmids for 24 h. After transfection, MG132 was added to the cells at 10 μM. Cell lysates were purified with anti-Flag beads, and proteins were analyzed by Western blotting. g Transfected HEK293 cells were treated with cycloheximide for the indicated times, and cell lysates were subjected to immunoblotting. Band intensities (mean ± SD, n = 3) on blots were analyzed using ImageJ and plotted. *P < 0.05 compared to control group.