Anti ship 1

The following anti-CD20 mAbs were used: the chimeric IgG1κ rituximab, the humanized IgG1κ obinutuzumab (GA101), and its non-glycoengineered parental molecule, GA101 wild type (WT), all kindly provided by Roche Innovation Center Zurich (Schlieren, Switzerland).
For functional assays, the following mAbs were used: anti-2B4 (clone:C1.7, #IM1607, Beckman Coulter Life Science), anti-NKp46 (clone: 9E2, #331902, Biolegend), anti-natural killer group 2 member D (NKG2D) (clone: 149810, #MAB139, R&D Systems), all mouse IgG1 isotype, and goat F(ab')₂ fragment anti-mouse IgG (H + L) (#115-006-003, Jackson ImmunoResearch Laboratories). The following fluorochrome-conjugated mAbs were used for flow cytometric analysis: anti-CD25 APC (clone:M-A2511, #555434) and anti-CD215 PE (clone:JM7A4, #566589) were from BD Biosciences; the anti-pS6 ribosomal protein (S235/236) PE (clone: D57.2.2E, #5316S) was from Cell Signaling Technology. For immunoblot analysis, antibodies were obtained from the following sources: anti-SHIP-1 (clone:P1C1, #sc-8425) from Santa Cruz Biotechnology Inc); the anti-phospho-STAT4 (Tyr693) (clone:D2E4, #4134), anti-STAT4 (clone:C46B10, #2653), anti-Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) (#4958) and anti-Akt (#9272), all from Cell Signaling Technology.

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.

Mice were perfused transcardially with 40 ml of cold PBS. Hemispheres were isolated and stored at −80 °C until analysis. Protein extraction and western blots were performed as previously described^{52 (link)}. Briefly, the whole-cell lysates were obtained by homogenizing in RIPA lysis buffer (Santa Cruz Biotechnology, Inc., sc-24948) and centrifuging (14,000 g at 4 °C for 30 min). Equal amounts of protein (50 mg) were loaded and subjected to electrophoresis on an SDS-PAGE gel. After being electrophoresed and transferred to a nitrocellulose membrane. To separate the region where the target protein will appear, the membrane was cut along the molecular weight marker. The member straps were then blocked and incubated with the primary antibody overnight at 4 °C. Following primary antibodies were used: anti-tryptase 1:1000 (Santa Cruz Biotechnology, sc-32889), anti-chymase 1:100 (Abcam, ab186417), anti- IL-1β (Abcam, ab9722) 1:750 and anti SHIP1 1:500 (Santa Cruz Biotechnology, sc-6244). The antibody against β-actin (Santa Cruz, 1:1000) was used as the internal control.
If the target protein had similar (+/− 20 kDa) weight compared to β-actin, the membrane straps were blocked and proceeded as described above.
Some representative strips were proceeded with “Microsoft Office 2010”