Detection of activation of cullin neddylation in DF-1 cells

To detect activation of cullin neddylation, DF-1 cells were seeded on six-well plates and cultured for 24 h, then treated with rMGA_0676 at concentrations of 0, 20, 40, or 80 μg/ml for 24 h. Then cell lysates were prepared for western blot to detect the expression of cull-NEDD8 based on the anti-chicken cull antibody or anti-NEDD8 antibody.β-actin was used as internal reference. DF-1 cells were seeded on six-well plates and cultured for 24 h, then treated with MLN4924 (500 ng/l) for 12–18 h or MGA_0676 at concentrations of 40 μg/ml for 24 h. Then cell lysates were prepared for western blot to detect expression of cull-NEDD8 and NF-κB activity as described above. β-actin was used as internal reference.