Figure 5

Detection and quantification of carbonyl groups on AAPH-treated 6PGDH samples. Solutions containing 6PGDH (58 μM monomer) with or without 10 or 100 mM AAPH were incubated at 37 °C in 75 mM phosphate buffer at pH 7.4. Aliquots were taken every 30 min up to 180 min, the AAPH was then removed, and samples kept at -80 ºC until analysis. Panel (A): quantification of the total carbonyl content on 6PGDH determined by DNPH assay incubated in the presence of 10 (open square) and 100 (open circle) mM AAPH. Panels (B) and (C): carbonyls detected by western blotting (using a commercial Oxyblot system) for samples exposed to 10 or 100 mM AAPH, respectively. On right vertical axis, black arrows indicate the band corresponding to 6PGDH monomers, while on left vertical axis, blue arrows indicate the corresponding molecular masses (obtained by comparison with molecular mass markers). For panels (B) and (C), it should be noted that images correspond to cropped and aligned WB images of membranes exposed for 50 s (as presented in Fig.S11). The data presented in panel (A) are the mean of at least three independent experiments, each developed in triplicate.