Phosphatase inhibitor cocktails 1 and 2

Immunoblotting was performed essentially as described (Győri et al., 2014 (link); Csete et al., 2019 (link)). Cells grown on 6-well tissue culture plates were washed with ice-cold PBS and lysed using radioimmunoprecipitation assay buffer (RIPA, containing 1% Triton X, 0.1% SDS, 0.5% sodium deoxycholate, 30 mM HEPES, 5 mM Na-EGTA, 10 mM benzamidine, and 20 mM sodium fluoride in physiological saline) supplemented with sodium-orthovanadate, phosphatase inhibitor cocktails 1 and 2, PMSF and aprotinin (all from Sigma). Cell debris was removed by centrifugation at 16,100 g. Protein concentration was determined using the Micro BCA^TM Protein Assay Kit (Thermo Fisher Scientific). Samples then were mixed with 4× reducing sample buffer and boiled for 10 min. Ten micrograms of total protein was run on a 14% SDS-polyacrylamide gel, electroblotted onto nitrocellulose membranes and stained with Ponceau. Membranes were then blocked with 3% dry milk in PBS and 0.1% Tween 20 (PBS-Tween), followed by immunoblotting with primary antibodies against eGFP (1:2,000; clone F56-6A1.2.3; Abcam) or β-actin (1:10,000; Clone AC-74; Sigma) diluted in 3% BSA in PBS-Tween, followed by peroxidase-labeled anti-mouse IgG antibodies (1:5,000; GE Healthcare) diluted in 3% dry milk in PBS-Tween. Signal was developed by ECL (GE Healthcare) and exposed to X-ray film.

Cells were lysed in radioimmunoprecipitation assay buffer (10 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with 1x complete inhibitor mix (Roche Diagnostics, Grenzach-Wyhlen, Germany) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich) and protein levels were analyzed by immunoblot using 30 μg of protein per lane mixed with Laemmli buffer containing β-mercaptoethanol, using the respective antibodies. Protein bands were visualized using horseradish peroxidase-coupled secondary antibodies (Sigma-Aldrich) and enhanced chemiluminescence (Perbio, Bonn, Germany).^{32 (link)}

Total protein and mRNA were simultaneously isolated from the gastric tissue samples using the AllPrep DNA/RNA/Protein Kit (Qiagen, Germany) according to the manufacturer's instructions. The protein pellet was dissolved in a buffer containing 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), 1% Protease Inhibitor Cocktail (Sigma) and 0.5% each of Phosphatase Inhibitor Cocktails 1 and 2 (Sigma-Aldrich, USA). The protein concentration was determined by the Bradford method (Sigma-Aldrich, USA). The RNA concentration and quality were determined using a NanoDrop spectrophotometer (Kisker, Germany), and the RNA integrity was determined by gel electrophoresis.

The cells were lysed by incubation with 1 mL lysis buffer (30 mM Tris, pH 8.5, 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM EDTA, 1 tablet of Complete Protease Inhibitor (Roche, Basel; Switzerland) per 100 mL, and 10 μL/mL of each of the phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich)) per 3 × 10⁷ cells for 10 min at 25°C. The resulting lysates were centrifuged for 30 minutes (14,000 ×g, 4°C) and stored at −80°C until further processing. Total protein concentration was determined by the 2D-Quant Kit (GE Healthcare, Uppsala, Sweden) according to the manufacturer's manual.

Blood was collected from four healthy volunteers at Wythenshawe MEU in cell preparation tubes (CPT; BD) and PBMCs were isolated by centrifugation within 2 h of collection. PBMCs were incubated for 1 h at 37 °C in RPMI culture media (Sigma) ± 30 nM AZD0156, then given 5 Gy irradiation using an X-Rad irradiator (Pxi). Cells were subsequently lysed in 6 M Urea, 25 mM Tris (pH8.0), 1 mM EDTA, 1 mM EGTA lysis buffer with phosphatase inhibitor cocktails 1 and 2 (Sigma) and protease inhibitors (Sigma). Lysate protein concentration was determined by BCA quantification (ThermoFisher). Lysates were reduced, alkylated with iodoacetamide, and digested with trypsin overnight at 37 °C. Formic acid was used to quench the reaction, a mix of stable isotope-labelled peptide standards were added prior to desalting using Oasis HLB 96-well plates and a positive pressure manifold (Waters).