Fig 2: The role of PDI in redox status of NMDAR.(A) Co-immunoprecipitation analyses of PDI interaction with IP3R, NR2A, NR2B, NR1 and M1R in the hippocampus. M, molecular weight marker. (B) The quantitative analyses of co-immunoprecipitation of PDI with NR1, NR2A, NR2B, M1R and IP3. PDI binds to NR1 and NR2A more than NR2B. (C) Schematics of modified biotin switch technique for the measurement of the amount of free thiols (-SH) and S-nitrosothiols (-SNO). (D) Representative western blot for the amount of -SH + -SNO in total protein extracts. M, molecular weight marker. (E–G) Effects of PDI siRNA, DTNB and bacitracin on thiols on NR1 and NR2. The amount of -SH + -SNO on NR1 and NR2A subunits are significantly reduced by PDI siRNA, DTNB and bacitracin. (E) Western blot representing the amount of -SH + -SNO on NR1 and NR2A subunit. M, molecular weight marker. (F,G) Quantification of effects of PDI siRNA, DTNB and bacitracin on the amount of -SH + -SNO on NR1 and NR2A subunit and PDI expression (mean ± S.E.M.; *p < 0.05 vs. control; n = 7, respectively).

Fig 3: Changed redox status of NMDAR by PDI in epileptic rats.(A) Western blot data for the amounts of -SH + -SNO of NR1 and NR2A subunits in non-SE (N), 3 day-post SE (3D), 1 week-post SE (1W) and chronic epileptic (6 week-post SE, Epil) animals. M, molecular weight marker. (B) Quantification of the amount of -SH + -SNO on NR1 and NR2A (mean ± S.E.M.; *p < 0.05 vs. normal; n = 7, respectively). The amounts of -SH + -SNO of NR1 and NR2A are increased 3 days after SE, and subsequently recovered to normal normal level 1 week after SE. (C) Representative photos for PDI expression in the normal and epileptic hippocampus. Bar = 300 µm. (D) Co-immunoprecipitation analyses of NR1 and NR2A interaction with PDI in normal and epileptic animals. M, molecular weight marker. (E) The quantitative analyses of co-immunoprecipitation of PDI with NR1 and NR2A in normal and epileptic animals (*p < 0.05 vs. normal; n = 7, respectively). In chronic epileptic animals, the amount of -SH + -SNO of NR1 is increased, as compared to normal animals.

Fig 4: Effect of PDI knockdown on spontaneous seizure activity in epileptic (6 week-post SE) rats.(A) Representative EEGs for control siRNA- and PDI siRNA-infused epileptic animals. (B–D) Anticonvulsive effect of PDI siRNA on spontaneous seizure activity: (B) the mean seizure frequency, (C) seizure duration and (D) behavioral seizure score (Open circles indicate each individual value. Closed circles indicate mean value. *p < 0.05 vs. control siRNA; n = 5, respectively). PDI knockdown inhibits the spontaneous seizure activity in chronic epileptic rats. (E) Co-immunoprecipitation analyses of NR1 or NR2A interaction with PDI in control siRNA- and PDI siRNA-infused epileptic animals. M, molecular weight marker. (F) The quantitative analyses of co-immunoprecipitation of PDI bound to NR1 and NR2A (*p < 0.05 vs. control siRNA; n = 7, respectively). (G) Western blot data for the amounts of -SH + -SNO on NR1 and NR2A subunits in normal (N), control siRNA- and PDI siRNA-infused epileptic animals. M, molecular weight marker. (H,I) Quantification of effects of PDI siRNA on the amount of -SH + -SNO on NR1 and NR2A subunit and PDI expression in epileptic rats (mean ± S.E.M.; *p < 0.05 vs. normal; n = 7, respectively). PDI siRNA infusion reduces the binding of PDI to NMDAR, the amount of -SH + -SNO of NMDAR subunits and PDI expression level in epileptic animals.