Rc dc protein assay

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.

Isolated islets were collected and washed twice with PBS (37 °C). Cells were suspended in the rehydration solution (7 M urea, 2 M thiourea, 4% CHAPS, 60 mM dithiothreitol (DTT), 0.002% bromophenol blue) added with 50 mM NaF, 2 mM Na₃VO₄, 1 μL/10⁶ cells protease inhibitors, 1 µM trichostatin A, 10 mM nicotinamide. After stirring and sonication (4 seconds, 5 times) cells were allowed to rehydrate for 1 h at room temperature (RT) with occasional stirring. Thereafter, the solution was centrifuged at 17,000 g for 5 min at RT. Protein concentration of the resulting supernatant was determined using the Bio-Rad RC/DC-protein assay (Bio-Rad). BSA was used as a standard. The global protein content was analyzed with focus on mitochondrial proteins.

The proteomic analysis was performed with islet preparations obtained from three different multiorgan donors (representing the biological replicates). Protein extraction from human pancreatic islets was performed as previously described [52 (link)]. Briefly, isolated islets were collected and washed twice with PBS (37 °C). Cells were suspended in the rehydration solution (7 M urea, 2 M thiourea, 4% CHAPS, 60 mM dithiothreitol (DTT), 0.002% bromophenol blue) containing 50 mM NaF, 2 mM Na₃VO₄, 1 μL/10⁶ cells of protease inhibitors, 1 µM trichostatin A, and 10 mM nicotinamide. After stirring and sonication (4 s, 5 times) cells were allowed to rehydrate for 1 h at room temperature (RT) with occasional stirring. Thereafter, the solution was centrifuged at 17,000× g for 5 min at RT. The protein concentration of the resulting supernatant was determined using the Bio-Rad RC/DC-protein assay (Bio-Rad). BSA was used as a standard.

Total protein for 2D-DIGE analysis was extracted as previously described [28 (link)]. Briefly, cells were washed three times in PBS and lysed in lysis solution (8 M urea, 2 M thiourea, 2.5% CHAPS, 2% ASB-14, 60 mM DTT, 40 mM Tris-HCl pH 8.8 and protease inhibitor cocktail) prior to sonication. Protein solutions were further purified by a modified TCA-acetone precipitation (2D-CleanUp Kit, GE Healthcare) and, finally, dissolved in the DIGE labelling buffer (8 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.0). Protein concentration was determined using the Bio-Rad RCDC Protein Assay (Bio-Rad, UK), following manufacturer’s instructions. Triplicate protein samples of each of the strains were labelled using Cy3 or Cy5 cyanine dyes and separated in three two-dimensional electrophoresis gels as previously described [28 (link)]. Fluorescence images of the gels were obtained on a Typhoon 9400 scanner (GE Healthcare). Cy3 and Cy5 images were scanned at excitation/emission wavelengths of 532/580 nm and 633/670 nm, respectively. Image analysis and quantification of relative protein abundances were performed using SameSpots (Nonlinear Dynamics, UK).