Thereafter, to clarify the mechanism by which NFκB/p65 may be involved in MLN4924-dependent inhibition of TNF-α-induced MMP9 gene expression in ESCC cells, the levels of phospho-NFκB/p65 (Ser536) and phospho-IкB-α (Ser32) were analyzed in whole cell lysates in the time–course and dose–response experiments by Western blotting (Figure 2C–E and Figure S5). This evidence concerns the gene MMP9 and esophageal squamous cell carcinoma.