A 769662

The in vitro preliminary kinase assays human α1β1γ1 AMPK were carried out according to the previous experimental method [33 (link)]. The screened compounds and α1β1γ1 AMPK isoform were provided by Topscience Co. (Shanghai, China) and Huawei Pharmaceutical Co. Ltd. (Shanghai, China), respectively. Generally, each of the evaluated compounds was dissolved in 10% Dimethyl sulphoxide at 10 μM and diluted to a required concentration with buffer solution. Then, 5 μL of the dilution was added to a 30 μL kinase assay buffer and 5 μL AMPK isoform per well. The solution was mixed at 0 °C for 30 min. Next, 5 μL of AMARA petide and 5 μL Adenosine triphosphate (ATP) were added to the well. The enzymatic reactions were conducted at 30 °C for 30 min. The AMPK activity was determined by quantifying the amount of ATP remaining in assay solution with Kinase-Glo Plus luminescent kinase assay kit (Promega, Madison, WI, USA). The luminescent signal is correlated with the amount of ATP present, while inversely correlated with the kinase activity. The mean values from three independent experiments were used for the expression of relative activities. A-769662, a β1-selective AMPK activator reported by Abbott laboratories, was used as a control.

Red blood cell-lysed mouse spleen cells were enriched for B cells using CD43 negative magnetic selection (Miltenyi). Cells were grown in RPMI1640 supplemented with 10% FBS and 50 μM β-mercaptoethanol. B cells were stimulated with 1 μg/ml anti-CD40 mAb (BD Pharmingen) and 25 ng/ml IL-4 (R&D Systems). At day 3, anti-CD40 was washed out and cells replated in medium containing only IL-4 until day 5. For early Ampk activation, phenformin (Sigma) was resuspended in 1x PBS, pH 7.4, and used at 100 µM, and A-769662 (Abbott) was resuspended in DMSO and used at 50 µM at the time of stimulation.