Active Thioredoxin-interacting Protein (TXNIP)

Thioredoxin-interacting Protein (TXNIP), a multifunctional member of the α-arrestin family, is tightly linked to cellular metabolism, redox balance, and disease pathogenesis. Initially identified as a binding partner of thioredoxin, it regulates glucose/lipid metabolism via a glucose-responsive negative feedback loop and acts as a tumor suppressor by inhibiting cell proliferation. TXNIP expression is dysregulated in diverse diseases, including type 2 diabetes, cancer, and retinal or cardiac disorders, with its upregulation often exacerbating oxidative stress and inflammation. It localizes primarily in the cytoplasm and nucleus, participating in both cytoplasmic signaling and nuclear gene regulation. TXNIP directly binds to NLRP3 to activate the inflammasome, triggering inflammatory cytokine release and cell pyroptosis.Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse TXNIP and recombinant human NLRP3. Briefly, TXNIP was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to NLRP3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TXNIP pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant mouse TXNIP and recombinant human NLRP3 was shown in Figure 1, the EC50 for this effect is 0.01193µg/mL.