FIG. 6.

Pulsatile NGF treatment triggers prolonged activation of protein kinase C, but not of MAPK. (A) Cell lysates were prepared from PC12 cells at the indicated times after either 2 min of pulsatile (pulse-chase) treatment or continuous treatment with 50 ng of NGF per ml. The proteins were electrophoresed and blotted onto nitrocellulose, and the blots were probed with anti-phosphotyrosine antibody. The positions of the ERK1 and ERK2 MAPKs are indicated. (B) At the indicated times after either 2 min of pulsatile (pulse-chase) treatment or continuous treatment with 50 ng of NGF per ml, PC12 cells were fractionated into cytosolic and membrane fractions by sonication and subsequent centrifugation. Membrane proteins were extracted, electrophoresed, and blotted, and the blots were probed with anti-protein kinase C-ɛ (anti-PKC ɛ) antibody (top) and with anti-N-CAM antibody (bottom). (C) Quantification was performed as described in Materials and Methods. The maximal response to continuous NGF treatment was given a value of 1.0.