Cells were treated with ANGII in 6-well plates with 200 μL of lysis buffer (0 .1M Tris-HCL, pH 7.5, 4% SDS, 0 .1M DTT) used for each well. The cells were collected with a cell scraper and vortexed 3 times for 5 min each. The lysates were sonicated with pulses of 30s on and 30s off, 20 times, at 4 °C and then incubated at 56 °C for 30 min. After centrifugation (16,000 x g for 5 min) supernatants were collected for digestion. The protein amount was measured using the Bio-Rad RC-DC protein assay, as instructed by the product manual. (Bio-Rad RC DC™ Protein Assay Kit II, 5000122). Total protein (12.5 μg) was digested with trypsin (trypsin, enzyme to protein ratio 1:20) and vortexed for 1 min and incubated at 37 °C for 16 h. After drying the peptide extract completely with a Speed Vac, 15 μL of 5% acetonitrile/0.1% formic acid was used to reconstitute the samples, which were then sonicated for 5 min. All contents were transferred to a new HPLC vial-Thermo #MSCERT5000-36LVW. A total of 6 μL was injected for LC-MS analysis [21 (link)]. Label-free quantification was obtained by MaxQuant 1.5.3.17. A database search was performed using PEAKS Studio 8.5.
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