The effects on phospho-ERK1/2 and ERK1/2 by GTX65641-pro Progranulin protein in neuronal differentiated mouse P19 cells. Undifferentiated mouse P19 cells were induced to differentiated in 1μM retinoic acid (RA) in α-minimum essential medium (αMEM) containing 10% heat-treated fetal bovine serum on bacterial grade plates for 3~4 days to allow aggregates to form (generation of embryonic bodies). The aggregates were then plated on tissue culture grade plates in the absence of RA for 3~4days. To examine the induction of signal of phospho-ERK1/2 and ERK1/2, reactions were carried out at 37ºC over 0, 5, 10, 30, 60, 120mins, respectively by adding the recombinant protein (500ng/ml) to the neuronal differentiated mouse P19 cells, which were maintained with serum starvation for 24hrs. Treatment with Progranulin protein (GTX65641-pro) was performed in lanes 1, 2, 3, 4, 5, and 6 over 0, 5, 10, 30, 60, 120mins, respectively. GAPDH was used as loading control for western blotting.

Deglycosylation of GTX65641-pro Progranulin (human) protein. To examine the deglycosylation of human Progranulin, 1 μg of human progranulin is denatured with 1X glycoprotein denaturing buffer at 100ºC for 10 minutes. After the addition of NP-40 and G7 reaction buffer, two fold dilutions of PNGase F are added and the reaction mix is incubated for 1 hour at 37ºC. Separation of reaction products is visualized by immunoblotting using anti-Progranulin (human) antibody.