Phosphatase inhibitor cocktail set 2 1

Western blotting was used for SBSN data validation in enriched serum (i.e., treated with ProteoMiner beads, as detailed above) and tissue, as previously described [45 (link)]. The control and EC tissues were lysed with 1% NP-40, 50 mM Tris-HCl (pH 8.0), NaCl 150 mM with Phosphatase Inhibitor Cocktail Set II 1× (Millipore, Burlington, VT, USA), 2 mM phenylmethylsulphonyl fluoride (PMSF), and 1 mM benzamidine.
In this study, 30 µg of protein from the tissue and the enriched serum were loaded on a 4–20% precast gel (Bio-Rad) and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% defatted milk in TBS-tween 20 after protein transfer and incubated overnight at 4 °C with 1:1000 diluted primary rabbit polyclonal antibody against SBSN (Abcam). After primary antibody incubation, membranes were washed three times with TBS-Tween 0.05% and incubated with HRP-conjugated anti-rabbit IgG and anti-mouse IgG (1:3000, Sigma-Aldrich; Merck Kagan, Darmstadt, Germany). The protein band signal was visualised using SuperSignal West Pico Chemiluminescent (Thermo Fisher Scientific Inc., Ottawa, ON, Canada). The intensities of the immunostained bands were normalised with the total protein intensities measured by staining the membranes from the same blot with a Red Ponceau solution (Sigma-Aldrich, St. Louis, MO, USA).

The phosphoprotein isolation from EC and healthy endometrium tissue samples was performed by Phosphoprotein Enrichment Kit (Pierce, Appleton, USA) [50 (link)]. About 5 mg of tissue from the EC or the healthy endometrium was homogenized in lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 8.0), NaCl 150 mM) with Phosphatase Inhibitor Cocktail Set II 1× (Millipore, Burlington, VT, USA) and 2 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine. Protein concentration was determined by Bradford assay. According to the manufacturer’s instructions, 3mg of tissue lysate was used for the phosphoprotein purification by immobilized metal affinity chromatography (IMAC).
3 EC was used to isolate phosphopeptides. Tissues were lysed, and proteins were digested with EasyPep Maxi MS Sample Prep Kit (Thermo Fisher, Boston, MA, USA). After protein digestion, peptides were transferred in dioxide (TiO₂) spin tips (Thermo Fisher) for phosphopeptide enrichment, according to the manufacturer’s instructions.