Hrp conjugated goat anti rabbit or anti mouse secondary antibodies

Protein extracts were prepared by lysing the macrophages for 5 min in boiling denaturing extraction buffer containing 1% SDS and 10 mM Tris (pH 7.4). The insoluble material was removed by centrifugation for 15 min at 12 000g at 4 °C. The total protein concentrations were determined using the BCA assay (Pierce/Thermo Fisher Scientific, Rockford, IL, USA). The cell extracts (standardized to 50 µg of total protein/lane) were separated by 12% denaturing SDS–PAGE and transferred to a PVDF membrane (Amersham Pharmacia Biotech, Freiburg, Germany) by semidry blotting using an electroblotter (Bio-Rad, Hercules, CA, USA) at 0.8 mA/cm² for 120 min. The membranes were blocked by 5% milk with PBST and incubated overnight with primary antibodies against total-Stat6, phospho-Stat6 (pSTAT6-Tyr641) and β-Actin (all from Cell Signaling Technology, Beverly, MA, USA) at 4 °C. After being washed in PBST three times, the membranes were incubated for 45 minutes with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in blocking buffer. The membranes were subsequently washed and incubated with ECL kits (Pierce/Thermo Fisher Scientific, Rockford, IL, USA). The signal intensity was quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA). The western blotting experiment was repeated at least three times.