Phosphatase inhibitor cocktail tablet

Cells were lysed in RIPA buffer containing a protease inhibitor mixture and a phosphatase inhibitor cocktail tablet (Roche Diagnostics, Basel, Switzerland). The protein concentration was analyzed by the bicinchoninic acid (BCA) assay kit (T-Pro Biotechnology, New Taipei City, Taiwan). Quantified protein sample (20 μg) was resolved on 7.5–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then proteins were transferred from the gel to polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked in 5% BSA solution for 1 h and incubated with the anti-RAGE (1:1000; Millipore) and PCNA (1:1000; Cell Signaling) antibodies overnight at 4 °C. The next day, the membrane was incubated with secondary anti-rabbit or anti-mouse antibodies (1:10,000; Cell Signaling) for 1 h at room temperature. The membrane was reacted with the Electrochemiluminescence (ECL) reagent and the visual signals were detected using a Luminescent Image Analyzer Amersham Imager 600 (GE Healthcare Life Sciences, MA, USA). The band densities were quantified using the Image-J software program.

For each subject, 16–18 mL of whole venous blood was collected in EDTA Vacutainers (BD Biosciences, Milan, Italy). Samples were processed within 40 min from collection to avoid degradation of phosphorylated proteins. PBMCs were isolated by centrifugation over the density gradient medium Lymphoprep™ (Axis-Shield, Oslo, Norway) and using SepMate^™-50 tubes (Stem Cell Technologies), according to the manufacturer’s instructions. 8 × 10⁶ PBMCs were stored at − 80 °C until protein extraction. PBMCs pellets were lysed on ice-cold lysis buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EGTA) plus protease inhibitor cocktail tablet (Roche, 05892970001) and phosphatase inhibitor cocktail tablet (Roche, PHOSS-RO). The homogenate was maintained on ice for 30 min and then centrifuged at 12.000 RPM for 25 min at 4 °C. The supernatant was collected and the protein concentration was measured in triplicate using the Bradford protein assay.

Cells were lysed in Kinexus lysis buffer: 20 mM MOPS, 5 mM EDTA, 2 mM EGTA, 30 mM NaF, 0.5% Triton X, 1 mM PMSF, pH 7.2, protease inhibitor (cocktail tablet, Roche; Basel, Switzerland), and phosphatase inhibitor (cocktail tablet, Roche). Samples (10–20 µg) were run on SDS/PAGE with NuPAGE precast 4–12% gradient gels (Invitrogen; Carlsbad, CA, United States) and blots were incubated overnight at 4°C with primary antibodies (Supplementary Table S1) followed by incubation for 1.5 h at room temperature (RT) with a species specific secondary HRP-conjugated antibody diluted 1:10000. Chemiluminescence detection was performed with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Bremen, Germany) on the LAS-3000 (Fujifilm Medical Systems Inc.; Stamford, Connecticut, United States). Relative protein expression levels were normalized to GAPDH and quantified using ImageJ software (NIH; Bethesda, MD, United States).

To determine their protein concentration, the ARPE-19 cells were lysed in immunoprecipitation assay buffer containing a phosphatase inhibitor cocktail tablet and a protease inhibitor mixture (Roche Diagnostics, Basel, Switzerland). The protein concentration was assayed using a bicinchoninic acid assay kit (T-Pro Biotechnology, New Taipei County, Taiwan). Quantified protein lysates (30 μg) were analyzed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resolved on 7%–15% polyacrylamide gels. The proteins were then transferred into polyvinylidene difluoride membranes. Each membrane was blocked in 5% BSA for 1.5 h and individually incubated with different primary antibodies against caspase 7, cleavage-caspase 7, Bax, Bcl-2, Cyt c, mTOR, p-mTOR (Ser2448), p-ULK1 (Ser757), ULK1, p62, LC3, Beclin-1, GAPDH, and β-actin at 4°C overnight. After being washed three times with Tris-buffered saline mixed with 0.05% Tween-20 (TBST), the membrane was incubated with secondary antimouse or antirabbit antibodies (1 : 10,000; Cell Signaling Technology, MA, USA) for 1 h at room temperature. The signals were detected using a luminescent image analyzer: the Amersham Imager 600 (GE Healthcare Life Sciences, MA, USA). Signal intensities were then quantified using ImageJ.