Western Blot Analysis of SHIP-1 Protein

Cell lysates were acquired as previously described [43 (link)]. Briefly, cells were pelleted and lysed in 1% NP-40, 10 mM Tris, 140 mM NaCl, 0.1 mM PMSF, 10 mM iodoacetamide, 50 mM Na fluoride, 1 mM EDTA, 0.4 mM Na orthovanadate, 10 μg/ml leupeptin, 10 μg/pepstatin, and 10 μg/ml aprotinin, followed by centrifugation to remove nuclei and cell debris. Protein concentrations for each sample were calculated using the Bradford Bio-Rad protein assay. Protein lysates (80 μg/sample) were separated by gel electrophoresis through a 10% SDS-PAGE gel and were transferred overnight at 20 V/hr to a PVDF membrane. The membrane was then probed with anti-SHIP-1 (Santa Cruz Biotechnology; clone P1C1) followed by horseradish peroxidase-conjugated anti-mouse-IgG (GE Healthcare Life Sciences). The blot was developed with a SuperSignal West Femto chemoluminescent reagent (Thermo Scientific). As a protein loading control, the blot was stripped and reprobed for β-actin.