FIG. 1.

Methylglyoxal induces NGF oligomerization in a dose- and time-dependent manner. (A) NGF was treated with vehicle (control; Ctl) or the indicated concentrations of MG for 72 h at 37°C, under sterile conditions. NGF was then analyzed by reducing SDS-PAGE followed by Western blot using NGF-specific antibodies. (B) NGF was treated with vehicle (Ctl) or MG (4 μM) for the indicated time (1–6 days) at 37°C, under sterile conditions. Samples were analyzed as indicated in (A). (C) NGF was treated with vehicle (NGF) or MG (500 μM; NGF-MG) for 72 h at 37°C, under sterile conditions. Samples were analyzed by size-exclusion chromatography. NGF control (black) eluted as a single peak corresponding to the dimer (D). NGF-MG (red) eluted as three peaks with an apparent molecular weight corresponding to the dimer (D), tetramer (T), and octamer (O). (D) Analysis of fractions collected in (C) by reducing SDS-PAGE followed by Western blot using NGF-specific antibodies. Noncovalent oligomerization is evidenced by the appearance of monomeric NGF (≈13 kDa) and lower order oligomers in the indicated fractions. MG, methylglyoxal; NGF, nerve growth factor