Figure 5. Prx IV forms oligomeric complexes in the ER.

A. NEM-treated lysates prepared from HT1080s over-expressing Prx IV were subjected to sucrose gradient fractionation. Lysates were fractionated under mild detergent (native) or denaturing conditions. An additional sample was fully reduced by treatment with 20 mM DTT and alkylation with NEM prior to denaturing gradient separation (denaturing + reducing). Following fractionation and TCA precipitation, SDS-PAGE was performed under non-reducing or reducing conditions (as indicated) and anti-Prx IV Western blotting undertaken. B. Organelle membranes were prepared from Prx IV over-expressing HT1080 cells and alkylated with NEM immediately (steady state) or following 15 minutes incubation with 5 mM H₂O₂. Contents were subsequently fractionated through sucrose gradients and TCA precipitated. Anti-Prx IV Western blotting analysis was carried out following SDS-PAGE under reducing conditions.