Fig. 4.

Both Collagen and ES can bind NGF present in medium, while laminin does not. (A) Schematic representation of NGF-depletion experiment. BSA or ES were coated directly on plastic or jointly with collagen or laminin for 24 h. Medium containing NGF was incubated in the presence of these substrates for 6 h; an aliquot of incubated medium was kept for ELISA, while the remaining amount was transferred to serum-starved PC12 medium and allowed to differentiate in it for 3 days. ES and collagen partially depleted NGF from incubation medium. Note the significant difference between collagen and ES for the formation of neurite-positive cells (B) and neurite formation (C). N=4, ^*P<0.05 and ^***P<0.01 versus plastic-treated group, ^##P<0.01, ^###P<0.001 in comparison between BSA and ES groups. (D) ELISA for NGF showed a significant decrease in NGF when medium was incubated in presence of ES or collagen. Note the quasi-absence of NGF depletion by laminin. N=4, ^*P<0.05 versus plastic group, ^##P<0.01 between BSA and ES. The presence of ES bound to collagen resulted in most of the inhibitory effect on PC12 cell differentiation as observed in quantifying neurite-positive cells (E) and neurite formation (F). N=4.