Alliance 4

For Western blot analysis, 100 μg of total kidney protein were separated on SDS-polyacrylamide minigels by electrophoresis (21 (link)). After transfer by electroelution to PVDF membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked for 1 h with 5% non-fat milk in Tris-buffered saline solution. Blots were then incubated overnight with primary antibodies for anti-VDR and anti-Klotho (1:500 for both; Santa Cruz Biotechnology, Santa Cruz, CA). The labeling was visualized with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit, 1:2,000, or anti-goat, 1:10,000; Sigma Chemical, St. Louis, MO) and enhanced chemiluminescence (ECL) detection (GE Healthcare Limited, Little Chalfont, UK). Kidney protein levels were further analyzed with a gel documentation system (Alliance 4.2; Uvitec, Cambridge, UK) and the software Image J for Windows (Image J-NIH Image). We used densitometry to quantitatively analyze the protein levels, normalizing the bands to β-actin expression (anti-β-actin, Sigma Chemical, St. Louis, MO).

Western blotting assays were performed to assess the expression of the following proteins: AQP4, NKCC1, Bcl-x, Bax and MnSOD. Cerebral tissue samples were run on 12% polyacrylamide minigels for AQP4, Bax, Bcl-x and MnSOD and on 8% polyacrylamide minigels for NKCC1. After transfer by electroelution to polyvinylidene fluoride membranes (Amersham Hybond-P; GE Healthcare Life Sciences, Little Chalfont, UK), blots were blocked with 5% non-fat milk and 0.1% Tween 20 in Tris-buffered saline for 1 hour. Blots were then incubated overnight with an anti-AQP4 antibody (1:2,000), NKCC1 (1:500), Bcl-x antibody (1:500), Bax antibody (1:500) and MnSOD antibody (1:200). The labelling was visualised with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit immunoglobulin G (IgG), diluted 1:2,000; anti-goat IgG, diluted 1:10,000; anti-mouse IgG diluted 1:2,000; or anti-mouse IgG, diluted 1:2,000, respectively; Sigma-Aldrich) using an enhanced chemiluminescence (ECL) detection system (Amersham ECL Western Blotting Detection kit; GE Healthcare Life Sciences). The ECL membranes were scanned using Alliance 4.2 (UVItec, Cambridge, UK), and the cerebral AQP4, NKCC1, Bcl-x and Bax protein expression levels were quantified using densitometry, normalizing the bands to actin expression.

For western blot analysis, 25 μg or 100 μg of total kidney protein were separated on 8%, 10% or 12% SDS-polyacrylamide minigels by electrophoresis [15 (link)]. After transfer by electroelution to PVDF membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline solution. Blots were then incubated overnight with primary antibodies for: Aquaporin 2 (AQP2, 1/1000), Sodium/phosphate cotransport type IIa (NaPi-IIa, 1/200), magnesium channel (TRPM6, 1/200), angiotensinogen (AGT, 1/100), angiotensin converting enzyme (ACE, 1/100) and α-Klotho (1/500). Primary antibodies were obtained from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). The labeling was visualized with horseradish peroxidase-conjugated secondary antibody (anti-rabbit, 1:2000, or anti-goat, 1:10000; Sigma Chemical, St. Louis, MO) and enhanced chemiluminescence (ECL) detection (GE Healthcare Limited, Little Chalfont, UK). Kidney protein levels were further analyzed with a gel documentation system (Alliance 4.2; Uvitec, Cambridge, UK) and the software Image J for Windows (Image J–NIH Image). We used densitometry to quantitatively analyze the protein levels, normalizing the bands to actin expression (anti β-actin, Sigma Chemical, St. Louis, MO).

For western blot analysis, 100 μg of total kidney protein were separated on SDS-polyacrylamide minigels by electrophoresis (15 (link)). After transfer by electroelution to PVDF membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline solution. Blots were then incubated overnight with primary antibodies for: TGF-β1 (1/500; Santa Cruz Biotechnology, Santa Cruz, CA); angiotensinogen (AGT) (1/200; Santa Cruz Biotechnology); α-Klotho (1/500; Santa Cruz Biotechnology); and VDR (1/500; Santa Cruz Biotechnology). The labeling was visualized with horseradish peroxidase-conjugated secondary antibody (anti-rabbit or anti-mouse IgG, 1:2000, or anti-goat, 1:10000; Sigma Chemical, St. Louis, MO) and enhanced chemiluminescence (ECL) detection (GE Healthcare Limited, Little Chalfont, UK). Kidney protein levels were further analyzed with a gel documentation system (Alliance 4.2; Uvitec, Cambridge, UK) and the software Image J for Windows (Image J–NIH Image). We used densitometry to quantitatively analyze the protein levels, normalizing the bands to actin expression (anti β-actin, Sigma Chemical, St. Louis, MO).

For Western blot analysis, 100 μg of total kidney protein were separated on SDS-polyacrylamide minigels by electrophoresis (16 (link)). After transfer by electroelution to PVDF membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked for 1 h with 5% non-fat milk in Tris-buffered saline solution. Blots were then incubated with primary antibodies for anti-eNOS (BD Bioscience, CA, USA); anti-iNOS (MyBiosource, CA, USA); anti-HO-1 (AssayDesigns, MI, USA); anti-MnSOD and anti-Nrf2 (Cayman Chemicals, MI, USA); anti-p47^phox and anti-p67^phox (Santa Cruz Biotechnology, CA, USA). The labeling was visualized with a horseradish peroxidase-conjugated secondary antibody (anti-mouse or anti-rabbit, Sigma Chemical, MO, USA) and enhanced chemiluminescence detection (GE Healthcare Limited, Little Chalfont, UK). Kidney protein levels were further analyzed with a gel documentation system (Alliance 4.2; Uvitec, Cambridge, UK) and the software Image J for Windows (Image J-NIH Image). We used densitometry to quantitatively analyze the protein levels, normalizing the bands to β-actin expression (anti-β-actin, Sigma Chemical, MO, USA).

Kidney samples were run on polyacrylamide minigels^{29 (link)}. After transfer by electroelution to polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK), blots were blocked with 5% nonfat dry milk in Tris-buffered saline. Blots were then incubated overnight with antibodies against aquaporin 2 (AQP2, 1:500), V1aR (1:500), NF-κB (1:500), and β-actin (1:100 000), all of which were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The labeling was visualized with a horseradish peroxidase-conjugated secondary antibody (anti-mouse, 1:4,000; Sigma Chemical, St. Louis, MO, USA) and enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA).
We scanned the enhanced chemiluminescence films with an imaging system (Alliance 4.2; UVItec, Cambridge, UK). We then used densitometry to perform a quantitative analysis of the antibodies, normalizing the bands to β-actin expression^{32 (link)}.

As previously described^{18 (link)}, kidney samples were run on polyacrylamide minigels. After transfer by electroelution to polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK), blots were blocked with 5% nonfat dry milk in Tris-buffered saline. Blots were then incubated overnight with antibodies against caspase 3 (1:500), heme oxygenase-1 (HO-1, 1:1,000), peroxiredoxin 6 (Prdx6, 1:1,000), and nitrotyrosine (1:1,000), all of which were obtained from Abcam (Cambridge, UK); and against glutathione peroxidase 4 (Gpx4, 1:1,000), obtained from Cell Signaling Technology (Danvers, MA, USA). We visualized the labeling with a horseradish peroxidase-conjugated secondary antibody, using enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). We scanned the films with an imaging system (Alliance 4.2; UVItec, Cambridge, UK), after which we used densitometry to perform a quantitative analysis of the antibodies employed, normalizing the bands to β-actin expression.