Enzyme linked immunosorbent assay kit

Control and diabetic rats (n = 6 per group) were either untreated (group C, group D) or diabetic rats treated with recombinant adenovirus expressing adiponectin (group APN, n = 6) or luciferase injected via tail vein for 1 week prior to tissue collection [29 (link)]. The increased expression level of adiponectin was confirmed by an enzyme-linked immunosorbent assay kit (AdipoGen Inc., Incheon, South Korea). The values of APN were expressed as micrograms per milliliter in plasma. Another group of diabetic rats (n = 6) were treated with a selective FoxO1 inhibitor AS1842856 (AS). AS1842856 has an IC50 of 0.033 mM to inhibit FoxO1 and can potently block FoxO1 at a final concentration of 0.05–1 mM without showing cytotoxicity [20 (link), 30 (link)]. Rats were administrated intragastrically with AS (100 mg/kg every time) or the same dose of β-cyclodextrin as solvent control 2 times a day with a 12-hour interval for a duration of 8 days before the rats were terminated. All rats were terminated at 5 weeks after induction of diabetes. Our preliminary study and/or former study showed that luciferase and β-cyclodextrin did not affect target proteins in the liver/plasma [31 (link)]; the respective groups were not shown in the result parts.

Serum adiponectin level was measured using a commercial enzyme-linked immunosorbent assay kit (Adipogen Corp., San Diego, CA, USA). This method had intra- and inter-assay coefficients of variations of ≤ 3.8 and ≤ 5.5%, respectively. The subjects were divided into the tertile (T1–T3) by serum adiponectin levels (Figure 1). The cut-offs at T1 and T3 were <6.041 μg/ml and ≥11.884 μg/ml, respectively. T3 was defined as high serum adiponectin level.