Fig. 2. Synaptic activity prevents the overoxidation of peroxiredoxins in response to an oxidative insult, and negatively regulates the Thioredoxin inhibitor Txnip.
A) Western analysis of Prx overoxidation using an anti-PrxSO2/3H specific antibody. Analysis normalized to appropriate Prx band intensity. *p<0.05 compared to control, H2O2-treated neurons (n=5). Note for upper (PrxIV) band, a higher exposure is often taken to more accurately assess loading (as is the case in the example shown). B,C) Thioredoxin, Thioredoxin reductase and 2-Cys Prx levels analyzed by Western blot after 24 h of the indicated treatments (n=3). D) q-RT-PCR of Txnip RNA from rat cortical neurons treated as indicated (this and all subsequent q-RT-PCR data are normalized to Gapdh levels. *p<0.05 compared to control (n=4)). E) Western analysis of Txnip protein expression in response to the indicated treatments (24 h, *p<0.05 compared to control, n=3). This and all quantitation of Western blots involves normalisation to β-tubulin or other suitable control (such as Akt in the case of Pi-Akt, Prx in the case of PrxSO2/3H). F) Co-immunoprecipitation of Thioredoxin with an anti-Txnip antibody in samples from neurons experiencing different levels of synaptic NMDAR activity for 24 h (*p<0.05, n=5). G) Thioredoxin activity (insulin-reducing assay) in neurons treated with MK-801, expressed as a % of the activity observed in BiC/4-AP-treated neurons (*p<0.05 compared to BiC-stimulated neurons, n=6).