Il 10

Protein lysates were subjected to 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes, respectively. After blocking with 5% skimmed milk for 2 h, membranes were incubated overnight at 4°C along with their respective primary polyclonal antibodies [adiponectin 1:500, leptin 1:1000, AMP-activated protein kinase (AMPK) 1:1000, PGC-1α 1:200, Abcam, United States; TNFα1:1000, IL-6 1:500, IL-10 1:1000, Abnova, Taiwan]. The membranes were then incubated with appropriate secondary horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulin G (IgG) antibodies at a 1:5000 dilution for 45 min at 37°C. Blots were visualized using an enhanced chemoluminescence (ECL) reagent (Beyotime, China) and quantified by scanning densitometry.

In order to examine the cytokines secreted by cells, enzyme-linked immunosorbent assay (ELISA) was performed to detect Flt3lig, B cell activating factor (BAFF), IL-2, IL-6, IL-10, LIF, SCF, IL-22, TNF-α, IL-15, IL-4 and IL-7 (Abnova Corporation, Abnova, USA) in the culture supernatant harvested after 72 hours of cultivation without FBS. The quantification of telomerase activity of cells was assayed using the telomerase PCR ELISA kit (Roche Diagnostics GmbH, Mannheim, Germany) based on the telomeric repeat amplification protocol (TRAP).