Fig. 2: The neddylation upregulates SREBP1c transcriptional activity and protein level.

a HEK293 cells were cotransfected with luciferase plasmid and the other plasmids listed, incubated for 48 h, and subjected to luciferase assay normalized relative to β-galactosidase activity. Data are expressed as means ± SD (n = 3). *P < 0.05 versus control group. b Transfected HepG2 cells were treated with MLN4924 (500 nM) and incubated for 24 h; luciferase activity was then measured. Results were normalized relative to β-galactosidase activity. c HepG2 cells were transfected with 50 nM siRNA for NEDD8 and treated with 500 μM OA for 18 h. RT-qPCR was performed to evaluate the expression of lipogenic genes. The mRNAs were quantified in reference to the 18S RNA levels, and presented as the means ± SD (n = 3). * denotes P < 0.05 versus the control group. d HepG2 cells were incubated with 500 μM OA and 250/500 nM MLN4924 for 18 h. Cells were subjected to RT-qPCR. e HepG2 cells were cotransfected with the Flag-SREBP1c and His-NEDD8 or His-NEDD8ΔGG plasmids for 48 h. Cell lysates were subjected to Western blotting to verify protein expression. f HepG2 cells were cotransfected with the indicated siRNAs (50 nM each) and Flag SREBP1c plasmid for 48 h. Proteins were analyzed by Western blotting. g HepG2 cells were transfected with the indicated siRNAs (50 nM each) for 24 h, and cells were treated with OA (500 μM) for a further 24 h. Nuclear extraction was performed in the cell lysates. Extraction samples were analyzed by Western blotting. h HepG2 cells were incubated with OA (500 μM), and 24 h later treated with MLN4924 for a further 24 h. Nuclear extraction was performed, and samples analyzed by immunoblotting.