Nah2po4

Dynamic analysis of cellular redox was carried out using four different live cell imaging dyes, that detect specific molecular species: cytoplasmic superoxide is analysed using DHE (Life Technologies, D11347); mitochondrial superoxide is detected with mitoSOX™ Red (Life Technologies, M36008); global cellular hydrogen peroxide (H₂O₂) and peroxyl radicals (HO₂) are measured using DCFDA (Sigma, D6883) while unbound reduced glutathione (GSH) is assayed with MCB (Life Technologies, M-1381MP). Cells were cells co-transfected with an appropriate fluorophore that would not interfere with the fluorescence of the dye. All dyes were administered to the recording medium (RM) onto cells plated on coverslips, within Attafluor® metal cell chambers (Molecular Probes™, Thermo fisher, A-7816). RM composition is: 5.6 mMKCl (Sigma, P9333), 10 mM D-(+)-Glucose (Sigma, G7528), 10 mM HEPES (Sigma, H4304), 4.2 mM NaHCO₃ (Sigma, 56297), 138 mM NaCl (Sigma, S5886), 2.6 mM CaCl₂ (Sigma, C7902), 1.2 mM NaH₂PO₄ (BDH,1024940), 1.2 mM MgCl₂ (BDH, 10149), pH 7.4.
DHE (10 μM), mitoSOX (10 μM), and DCFDA (15 μM) were diluted in RM and imaged onto a Zeiss LSM510 confocal microscope. MCB was used at final concentration of 2.5 μM in RM. Brightness controlling settings were maintained consistently within the experiment for all techniques.

For western blot analysis, cells or kidneys were lysed in lysis buffer solution of 150 mM NaCl (Sigma-Aldrich, #s9625), 20 mM Na₂HPO₄ (BDH, #10494 L)/NaH₂PO₄ (BDH, #102455 S), 10% glycerol (Sigma-Aldrich, #G7757), 1% Triton X-100 (pH 7.2), complete protease inhibitor cocktail (Roche, #11836145001) and phosphatase inhibitors (1 mM final concentration of glycerophosphate (Sigma-Aldrich, #G9891), sodium orthovanadate (Sigma-Aldrich, #S6508) and sodium fluoride (Sigma-Aldrich, #S6521)). Total lysates were then quantified with Bio-Rad Protein Assay Dye reagent (Bio-Rad Laboratories, #500-0006), and Laemmli buffer at a final concentration of 2× was added to the samples. Proteins were next resolved in 4–12% Tris–glycine gradient gels (Life Technologies, #NP0335BOX) and then transferred onto Immobilon-P polyvinylidene fluoride membranes (Millipore, #IPVH00010). Membranes were blocked with 5% milk in Tris-buffered saline, Tween 20 (Sigma-Aldrich, #P1379) (TBS-T). All the primary antibodies for western blot analysis were diluted in 3% BSA in TBS-T. HRP-conjugated secondary antibodies were diluted 1:10,000 in 5% milk, TBS-T, and detection was performed with ECL (GE Healthcare, #RPN2106) alone or supplied with 10% SuperSignal West Femto (Thermo Fisher Scientific, #34095) when necessary.