TXNIP induction is evident in microarrays using both total and polysome-associated mRNA. From parent INS-1 cells exposed to 1 μM Tg, northern blots and Q-PCR show that total TXNIP mRNA increases by ~10-fold within 2 hours (Figure 1F and G). Treatment with the ER stress agent tunicamycin (Tm), an inhibitor of N-linked glycosylation, also increases TXNIP mRNA (Figure 1H). Validating our immunoaffinity purification strategy, we fractionated ribosomes according to size, and found, using yet another mediator of ER stress, the anterograde protein trafficking poison Brefeldin A (BFA), an approximately 50-fold migration of TXNIP mRNA from monosomes (in the uninduced state) to polysomes (Figure 1I).
