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Polydatin promotes Nrf2-ARE anti-oxidative pathway through activating Sirt1 to resist AGEs-induced upregulation of fibronetin and transforming growth factor-1 in rat glomerular messangial cells

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Resuspend the cell pellet in 100 L ultrapure water, mix well, take 50 L of the suspension, add 200 L pre-cooled methanol, vortex at 2500 r/min for 2 min, freeze the sample in liquid nitrogen for 5 min, thaw on ice for 5 min, vortex for 2 min, repeat this freeze-thaw-vortex cycle 3 times, centrifuge at 12,000 r/min, 4 C for 10 min, transfer 200 L supernatant to a new tube, store at 20 C for 30 min, centrifuge again under the same conditions, take 180 L supernatant, filter through a protein precipitation plate, analyze using UPLC (Waters ACQUITY H-Class D) and MS/MS (QTRAP 6500+), prepare standard solutions, construct a database from chromatographic peak intensities, perform qualitative analysis using MultiQuant 3.0.3 software, for the remaining 50 L suspension, repeat the freeze-thaw process 3 times, centrifuge, determine protein concentration by the BCA method, and use protein concentrations of parallel samples to calibrate metabolomics data

Mulcahy C, Mohamed AS, Weber R, Lai S, Fuller C, Rosenthal D, El-Naggar A, Hutcheson K, Gunn B, Garden A, Kies M, Lewin J, Morrison W, Zafereo ME