Fig. 5. Activation of GSK3ß by a soluble factor from differentiated PC12 cell extracts. (A) Equal amounts of protein from cells exposed to NGF for zero or five days were depleted of GSK3ß using specific antibodies (IPS, 0 DIV or 5 DIV). The extracts were collected and used in kinase assays alone or in conjunction with undepleted fractions from cells exposed to NGF for zero or five days (S1, 0 or 5 DIV). The in vitro kinase activity towards recombinant MAP1B present in the resultant samples was determined by immunoblotting with mAb SMI-31 (1B750-P); recombinant protein was detected with anti-GST serum (1B750). GSK3ß levels were determined by immunoblotting samples of the input fractions. Note the absence of kinase activity in differentiated cell extracts immunodepleted of GSK3ß (IPS (5 DIV)) and undifferentiated cell extracts (S1 (0 DIV)). Undifferentiated cell extracts mixed with immunodepleted differentiated cell extracts show high activity, but no significant increase in GSK3ß level. By contrast, immunodepleted undifferentiated cell extracts (IPS (0 DIV)) do not activate phosphorylation of MAP1B. (B) Prior phosphorylation of substrate (priming) does not activate MAP1B phosphorylation by GSK3ß. 1B750 was incubated with differentiated cell extracts immunodepleted of GSK3ß (IPS (5 DIV)) or with complete differentiated cell extracts in the presence of 20 mM LiCl for 16 hours at 37°C (S1 (5 DIV)/Li⁺) – conditions that inhibit phosphorylation of MAP1B by GSK3ß – and washed extensively in kinase buffer (at least 20 times the bead volume). The washed 1B750 was incubated with control buffer or with differentiated cell extracts (S1 (5 DIV)) or undifferentiated cell extracts (S1 (0 DIV)) for a further 16 hours at 37°C and kinase activity was then determined as in (A). Note that undifferentiated cell extracts show no kinase activity.