Complete set of protease inhibitors

Cells were lysed in ice-cold RIPA buffer containing 2 mM Na₃VO₄, 10 mM NaF and a complete set of protease inhibitors (Roche Diagnostics, Mannheim, Germany). The protein concentration was determined using the DC Protein Assay Reagent (Bio-Rad, Milano, Italy). Protein aliquots (60 μg for each condition) were incubated overnight at 4 °C with the anti-D1R antibody (1:40 dilution) in RIA buffer (400 mM NaCl, 20 mM EDTA, 20 mM Na₂HPO₄) containing 0.1% SDS. Protein-A agarose beads (Santa Cruz Biotechnology Inc., Heidelberg, Germany) were added, and incubation was continued for 3 h at RT. The beads were collected and extensively washed with a buffer containing 1% NP-40, 10% sodium dodecyl sulfate (SDS), 5% sodium deoxycolate and 10 μg/mL phenylmethylsulfonyl fluoride (PMSF) in PBS. The resulting proteins were analyzed using WB with the anti-Shp2 (1:1000) antibody.

Placental tissue processing was performed as described by Sammar et al. [31 (link)]. Briefly, placental tissue was homogenized and solubilized in RIPA lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 20 mM Tris/HCl (pH 8.0), 150 mM NaCl) containing a complete set of protease inhibitors (Hoffman la Roche, Switzerland) for 30 min at 2–8 °C. Insoluble material was removed by centrifugation and protein concentration was determined by BCA reagent (Pierce, Rockford, IL, USA). Placental lysate samples were aliquoted and stored at −70 °C until use.
For Western blot analysis, 50 µg of total protein lysates was separated on 12.5% SDS-PAGE and electro-transferred to a nitrocellulose membrane. After blocking free binding sites with 5% non-fat milk in 20 mM Tris/HCl buffer at pH 8.0 supplemented with 150 mM NaCl, membranes were probed with the anti-CD24 mAb SWA11 and anti-β-actin antibodies (0.1 µg/mL) overnight at 4 °C. Bound immune complexes were detected by horseradish peroxidase-conjugated rabbit anti-mouse IgG and developed by ECL detection kit (Biological Industries, Beit Haemek, Israel). β-Actin was used as reference housekeeping protein for equal protein loading and densitometry analysis. Signals were developed by chemiluminescence and were captured by an imager (Bio-Rad, Hercules, CA, USA).