Irf 1

Cells were harvested by centrifugation at 120 g for 5 minutes in a microfuge. The pellets were washed with PBS and lysed with mammalian protein extraction reagent (Pierce) with protease and phosphatase inhibitors (Sigma). The lysates were frozen at −80°C overnight and defrosted on ice, then cleared by centrifugation at 16 060 g for 10 minutes at 4°C. The supernatants were collected, and protein concentrations were determined with the Pierce BCA Protein Assay (Thermo Fisher Scientific). Equal amounts of total protein lysates (30 μg) were resolved by SDS‐PAGE, electrotransferred onto PVDF membranes, probed with antibodies, and detected with ECL reagent (GE Healthcare). The antibodies used for western blots are as follows: IRF‐1 was from BD Biosciences (San Jose, CA); GAPDH, c‐Raf, Lyn, Fgr, p47phox, Slp‐76, Vav1, PU.1, horseradish peroxidase anti‐mouse, and anti‐rabbit antibodies were from Cell Signaling (Danvers, MA); and c‐Cbl was from Santa Cruz Biotechnology (Santa Cruz, CA). The relative intensity of specific band was calculated against GAPDH using ImageJ software.