Progranulin (human), (recombinant)

Figure 2: Deglycosylation of Progranulin (human) (rec.) (Prod. No. ALX-201-377).Method: To examine deglycosylation of Progranulin (human) (rec.) (Prod. No. ALX-201-377), 1 µg of Progranulin (human) (rec.) (Prod. No. ALX-201-377) was denatured with 1x Glycoprotein Denaturing Buffer at 100°C for 10 minutes. After the addition of NP-40 and G7 Reaction Buffer, two-fold dilutions of PNGase F were added and the reaction mix was incubated for 1 or 3 hour at 37°C. Separation of reaction products was visualized by immunoblotting with Polyclonal Antibody to Progranulin (human) (Prod. No. ALX-210-465).  Lane 1: non-treatedLane 2: PNGase F, 1h Lane 3: PNGase F, 3h

Figure 3: The effects of phospho-ERK1/2 and non phospho-ERK1/2 by Progranulin (human) (rec.) (Prod. No. ALX-201-377) in THP-1 cells. Method: To examine the signal of phospho-p44/42 MAPK and p44/42 MAP kinase, reactions were carried out at 37°C over 0, 30, 60, min., respectively by adding the recombinant protein (100ng/ml) to the THP-1 monocyte cells, which were maintained with serum starvation for 24 hours. Recombinant proteins in lanes 1, 2 and 3 were subjected to THP-1 monocyte cell treatments over 0, 30, 60 min., respectively.

Figure 4: The effects of phospho-ERK1/2 and non phospho-ERK1/2 by Progranulin (human) (rec.) (Prod. No. ALX-201-377) in MCF10A cells. Method: To examine the signal of phospho-p44/42 MAPK and p44/42 MAP kinase, reactions were carried out at 37°C over 0, 30, 60, min., respectively by adding the recombinant protein (500ng/ml) to the MCF10A human breast epithelial cells, which were maintained with serum starvation for 24 hours. Recombinant proteins in lanes 2, 3 and 4 were subjected to MCF10A human breast epithelial cell treatments over 0, 30, 60 min., respectively.

Figure 5: Chemotaxis effect of Progranulin (human) (rec.) (Prod. No. ALX-201-377) in THP-1 cells.Method: Cell migration of THP-1 monocytic leukemia cells ws evaluated in disposable 24-well transwell polystyrene membrane with 8µm size pores. 0ng/ml, 10ng/ml, 100ng/ml and 250ng/ml of human recombinant progranulin were diluted in RPMI 1640 supplemented with 0.5% FBS respectively. In case boiled human progranulin, proteins were boiled at 100°C for 10 min. and then human progranulin samples were added to the lower chamber. THP-1 cells (2x10⁵ cells per well) in the same media with human recombinant progranulin (Prod. No. ALX-201-377) were added to the upper chamber. After 16 hours incubation at 37°C in 5% CO₂ humidified atmosphere, migration cells in the lower chamber were counted. Migrated cells in two separate fields per well from triplicate wells were enumerated on a hemacytometer by means of light microscopy.

Figure 6: Transwell migration assay comparing Progranulin (human) (rec.) (Prod. No. ALX-201-377) (hPrgn) and human monocyte chemoattractant protein-1 (hMCP-1) using PTX-treated or PD98059-treated THP-1 cells.