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). Here, TNF is linked to esophageal squamous cell carcinoma.