Western Blot Analysis of Cell Signaling

Single-cell suspensions of BM, digested pancreatic tumors cells and UN-KC-6141 cells treated with API (40 µM) were lysed with radioimmunoprecipitation buffer (Sigma-Aldrich), quantified with a BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA), resolved by SDS-PAGE (Thermo Fisher Scientific) and transferred onto a nitrocellulose membrane as previously described [12 (link)]. Membranes were probed with either anti-SHIP-1 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-iNOS (Cell Signaling Technology) or anti-Bcl-2 (Cell Signaling Technology, Danvers, MA, USA), all at a dilution of 1:1000. All blots were re-probed with a housekeeping protein, HRP-conjugated anti-β-actin (Sigma-Aldrich), at a dilution of 1:20,000. Membranes were then probed with the respective secondary anti-mouse or rabbit IgG HRP-conjugated antibodies (1:1000) of SHIP-1 and iNOS. Detection of SHIP-1, iNOS and β-actin proteins was performed using Super Signal West Pico or Femto Chemiluminescent Substrates (Thermo Fisher Scientific) and then imaged on a Bio-Rad Chemi Doc XRS Imaging System. Normalized densitometric ratios (divided by β-actin) were determined by quantifying WB results using Image J.