Progranulin (human) (rec.) (untagged)

Figure 2: Deglycosylation of Progranulin (human) (rec.) (untagged) (Prod. No. AG-40A-0188Y). To examine the deglycosylation of human Progranulin, 1 μg of human progranulin is denatured with 1X glycoprotein denaturing buffer at 100°C for 10 minutes. After the addition of NP-40 and G7 reaction buffer, twofold dilutions of PNGase F are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by immunoblotting using anti-Progranulin (human), pAb (Prod. No. AG-25A-0112).

Figure 3: The effects on phospho-ERK1/2 and non-phospho-ERK1/2 by Progranulin (human) (rec.) (untagged) (Prod. No. AG-40A-0188Y) in neuronal differentiated mouse P19 cells. Undifferentiated mouse P19 embryonal carcinoma 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-p44/42 MAPK and p44/42 MAP kinase, reactions were carried out at 37°C over 0, 5, 10, 30, 60, 120mins, respectively by adding the recombinant protein (500ng/ml) to the neuronal differentiated mouse P19 embryonal carcinoma cells, which were maintained with serum starvation for 24hrs. Treatment with Progranulin (human) (rec.) (untagged) 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.

Figure 4: Regulation of food intake and body weight by Progranulin (human) (rec.) (untagged) (Prod. No. AG-40A-0188Y). Permanent 26-gauge stainless steel cannulae were implanted into the third ventricle (ICV), or into the bilateral mediobasal hypothalamus (iMBH) of mice. After a 1-week recovery period, mice were handled daily for 3 days to acclimatize them to the injection procedure. Correct positioning of ICV-implanted cannulae was tested by verifying the presence of a dipsogenic response to angiotensin-2 (50 ng). The correct positioning of each iMBH cannulae was confirmed by histological examination, performed by independent observer after each animal was sacrificed. Only mice in which cannulae had been correctly positioned were included in data analysis. The compounds 2-DG and AICAR were dissolved in 0.9% (w/v) saline and administered in a total volume of 2.5 ml for ICV injection and 1 ml for iMBH injection, respectively. Food intake and body weight were monitored for 24 h post-injection. *See reference 1.

Figure 5: Progranulin (human) (rec.) (untagged) (Prod. No. AG-40A-0188Y) binds to the recombinant human receptor Sortillin much stronger than the Flag-tagged version of Progranulin (Prod. No. AG-40A-0068Y).
Method: Sortilin (human) (rec.) (His) (Prod. No. AG-40B-0229) is coated on an ELISA plate at 1 μg/ml overnight at 4°C. Progranulin (human) (rec.) (untagged) (Prod. No. AG-40A-0188Y) or the Flag-tagged version of Progranulin (Prod. No. AG-40A-0068Y) is added (starting at a concentration of 100nM with a twofold serial dilution) during one hour at RT and the interaction is then detected using a biotinylated anti-human Progranulin.