Rabbit anti ho 1

For immunoblot analysis, total protein was extracted by RIPA lysis buffer (Beyotime, China), and centrifuged at 12,000 g for 15 min at 4°C. Then, equal amounts of protein samples (30 μg per lane) from kidney or HK-2 cells were resolved on a 10–12% SDS-PAGE gel and then transferred onto PVDF membranes (Millipore) after electrophoresis. After blocking with 5% non-fat milk for 3 h at room temperature, the PVDF membranes were washed with TBST and then incubated overnight at 4°C with the following antibodies: rabbit anti-Nrf2 (1:1000, Abcam); rabbit anti-HO-1 (1:5000, Abcam); rabbit anti-NQO-1 (1:5000, Abcam); rabbit anti-cleaved-caspase3 (1:1000, CST); rabbit anti-cytochrome c (1:1000, CST); rabbit anti-Bax (1:1000, CST); rabbit anti-Bcl-2 (1:1000, CST); rabbit anti-PARP (1:1000, CST); rabbit anti-caspase 9 (1:1000, CST); and mouse anti-β-actin (1:5000, Sungene Biotech, Tianjin, China). Subsequently, the membranes were washed three times with TBST for 30 min and incubated with the appropriate secondary horseradish peroxidase (HRP)-conjugated antibodies for 1 h at room temperature. Immunoreactive bands were visualized by an ECL chemiluminescence system (GE Healthcare, Piscataway, NJ, United States), and densitometric analysis of the bands was performed using Quantity One software.

Neural stem cells were lysed in 50 mM Tris–HCl, pH 7.4, containing 100 mM NaCl, 1 mM MgCl₂, 0.1 mM CaCl₂, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (plus phosphatase and protease inhibitors) (Sigma-Aldrich). Proteins were separated by SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences Corp., United Kingdom). Antibodies were diluted in 0.1% Tween buffer (+ 5% BSA) as follows: rabbit anti-Nrf2 (1:500, Abcam, United Kingdom), mouse anti-NQO1 (1:2000, Novus Biologicals, United States), rabbit anti-HO-1 (1:2000, Abcam), mouse anti-Tubulin (1:1000, Sigma-Aldrich), rabbit anti-Frataxin (1:1000, Santa Cruz Biotechnology Inc., TX, United States). Signals were detected by enhanced chemiluminescence (ECL) (BioRad, CA, United States).

Biopsies from two aHUS patients - P1 carrying C3 p.R161W [C3 gain of function (9 (link))] and P2, having homozygous FH mutation p.C564F (FH deficiency)—were retrieved from the archive of the Pathology Institute of CHU Lille, France. P1 had a hemolytic anemia and histological analysis described typical lesions of glomerular and arteriolar TMA. Hemolysis was corrected at the moment of P2 biopsy, which reported predominant chronic TMA lesions, characterized by vessel wall thickening and thrombotic occlusion of many arterioles. Perls' Prussian blue staining identified hemosiderin deposits. Immunohistochemical analysis for HO-1 (rabbit anti-HO-1, Abcam) was performed on deparaffinized slides using Ventana XT autostainer (Ventana Medical systems). Immunofluorescence with rabbit anti-C3c (Dako) was performed on frozen slides. Secondary antibodies were coupled to Fluorescein IsoThioCyanate—FITC (Sigma Aldrich). A normal protocol, kidney allograft biopsy performed 3 months after transplant was used as a negative control and biopsies of two patients with chronic hemolysis (hemolysis associated with prosthetic heart valve) were used as positive controls for hemosiderin detection and tubular staining of HO-1 (20 (link)). Whole slides were scanned or analyzed by an Olympus microscope (Life Sciences Solutions) or Nanozoomer (Hamamatsu).

Protein concentrations were determined with the BCA Protein Assay Kit (Thermo Fisher Scientific, United States).The sample protein 25 μg was added into lysate to denaturate, and then electrophoresis by SDS-PAGE, transferred to PVDF membranes. After being blocked with 5% skimmed milk for 1 h, the membranes were incubated with primary antibodies overnight on a shaker. The primary antibodies (Abcam, Cambridge, UK), including rabbit-anti-Nrf2 (diluted at 1: 1000), rabbit-anti-HO-1 (diluted at 1: 1500), rabbit-anti-NQO-1 (diluted at 1: 1500), rabbit-anti-β-actin, and rabbit-anti-GAPDH (diluted at 1:3000), were used as a loading control. After being washed with Tris-bufered saline, the membranes were incubated with horseradish peroxidase conjugated anti-rabbit secondary antibody (diluted at 1:1500, Abcam, Cambridge, UK). Finally, Image J software (NIH, Bethesda, MD, USA) was used to analyze the band density of western blots.

Western blotting was conducted as previously described [14 (link)]. Briefly, periventricular brain tissue (~ 1 mm thick around the ventricles) was sampled and sonicated in Western sample buffer. Bio-Rad protein assay kit was used to equalize the protein amount in each sample. Samples were then separated with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a Hybond-C pure nitrocellulose membrane (Amersham, Pittsburgh, USA). The primary antibody was rabbit anti-HO-1 (1:2000 dilution; Abcam). To visualize the antigen–antibody complex, the ECL chemiluminescence system (Amersham) and a Kodak X-OMAT film were used. The image was analyzed by Image J software to determine relative densities.

Rats were euthanized using pentobarbital (100 mg/kg, intraperitoneal) and perfused intravascularly with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4). Brains were harvested and sectioned into 18-μm-thick slices with a cryostat after embedding. Immunohistochemical and immunofluorescence studies were performed as previously described [13 (link)]. The primary antibodies were rabbit anti-HO-1 (1:400 dilution; Abcam, Cambridge, USA), goat anti-Iba-1 (1:400 dilution; Abcam), mouse anti-CD68 (1:100 dilution; Abcam), mouse anti-rat CD163(1:100 dilution; AbD Serotec, Hercules, USA), polyclonal rabbit anti-alpha smooth muscle actin (1:200 dilution; Abcam). The secondary antibody in the immunofluorescence studies was Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen, Carlsbad, USA). Nuclear labeling was performed using fluoroshield™ with DAPI (F6057). Negative controls were performed without primary antibodies.

The endothelial cells onto the coated slide were fixed with 4% paraformaldehyde (PFA) for overnight at 4°C. After washing with PBS for three times, cells were blocked with 5% BSA for 1 h at room temperature. After washing with PBS, cells were reacted by the rabbit anti-PI3K (Santa Cruz Biotechnology), rabbit anti-pAkt (1:1000, Cell signaling), rabbit anti-Nrf-2 (1:1000, Santa Cruz Biotechnology), rabbit anti-HO-1 (1:1000, Abcam) and goat anti-Cox-2 (1:500, Santa Cruz Biotechnology) respectively at 4°C for 1 day, followed by FITC or Rhodamine-conjugated secondary antibody (1:1000, Jackson ImmunoResearch) at room temperature for 2 h. After washing with PBS, stained cells were mounted with Vectashield with DAPI (Vector Lab). Slides were observed under LSM 710 confocal laser scanning microscope (Carl Zeiss).