Neutrophil Protein Isolation and Western Blot Analysis

Protein isolation from cultured neutrophil was performed as previously described [16 (link)]. Western blots were performed using the standard SDS-polyacrylamide gel electrophoresis method and enhanced chemiluminescence detection reagents (GE Healthcare Biosciences AB, Uppsala, Sweden). Antibodies against phosphorylated signal transducer and activator of transcription 1 (pSTAT1) (Multi-clonal, Abcam, Cambridge, USA), STAT1 (Multi-clonal, Cell signaling technology, Beverly, MA), SOCS1 (Multi-clonal, Abcam, Cambridge, USA), and GAPDH (Cell signaling technology, Beverly, MA) were used. Immunoreactivity was semi-quantitatively measured by gel densitometric scanning and analyzed by the MCID image analysis system (Imaging Research, Inc.).