Phosphorylated stat3

The proteins were extracted from the cell pellets. Proteins were boiled for 10 min and loaded onto 4–12% bis Tris gels (Invitrogen, Waltham, MA, USA) for separation. The separated proteins were then blotted onto polyvinylidene difluoride (PVDF) membranes (Invitrogen) with 20% (v/v) methanol in NuPage Transfer Buffer (Invitrogen) at 15 V for 4 h at 4°C. Tris-buffered saline containing 0.01% Tween 20 (TBST) in 5% skim milk (Difco, BD Biosciences, Oxford, UK) was used to block the membranes for 1 h. The blots were washed three times with TBST for 10 min and then incubated overnight at 4 °C with primary antibodies specific to the following target proteins: phosphorylated JAK1, phosphorylated JAK2, phosphorylated STAT1 (1:1000; Cell Signaling Technology, Cambridge, UK), CSF1, IL-6, PTPN6, RAC2, TNFα, IL-1α, IL-1β, phosphorylated STAT3, STAT3 ADORA2A, JAK1, JAK2, STAT1, SOCS3, Bax, Bcl-2, and β-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation, the blots were washed thrice with TBST and incubated for 1 h with a horse-radish peroxidase–conjugated secondary antibody (1:3000; Santa Cruz) at 25°C. Finally, the blots were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK).