Isolation and Analysis of Granulocyte Proteins

Cytoplasmic and nuclear lysates were prepared as previously described [24 (link)]. To prepare total cell lysates from granulocytes, the cells were precipitated in 10% trichloroacetic acid (Wako) for 30 min on ice. The TCA-precipitated fraction was treated with a lysis solution containing 9 M urea, 2% Triton X-100, and 1% dithiothreitol, and was disrupted by ultrasonication, followed by an addition of 2% lithium dodecyl sulfate and further ultrasonication. The proteins were separated on a 10% polyacrylamide gel, transferred onto Immobilon-P Transfer Membranes (Millipore, Billerica, MA, USA), and blotted with rabbit polyclonal anti-c-Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit polyclonal anti-actin (Sigma), followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz). Signals were detected by ECL Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK).