Sc 24948

Western blotting was performed as previously described [5 (link)]. In brief, rats were anesthetized with isoflurane at the established time points after ICH and transcardially perfused with cold PBS. The right brain hemisphere was homogenized in RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology, USA) and further centrifuged at 14000 rpm for 30 min. Equal amounts of protein (4 μL, 30 μg) were loaded onto a 10% SDS-PAGE gel. After separation, the proteins were transferred to a nitrocellulose membrane, which was further blocked for 2 h with a blocking solution. The membranes were incubated with the following primary antibodies overnight at 4°C: anti-albumin (1 : 500, ab207327, Abcam, USA), anti-p-ERK (1 : 1000, Santa Cruz Biotechnology, USA), anti-ERK (1 : 1000, sc-514302, Santa Cruz Biotechnology, USA), anti-Nrf2 (1 : 1000, GTX103322, Gene Tex), anti–HO-1 (1 : 1000, ab68477, Abcam, MA, USA), anti-Romo1 (1 : 1000, Aviva Systems Biology, San Diego, CA), anti-Bcl2 (1 : 1000, ab59348, Abcam, MA, USA), and anti-Bax (1 : 1000, Littleton, CO). The membranes were incubated with an anti-β-actin antibody (1 : 5000, sc-47778, Santa Cruz Biotechnology, TX, USA).) The results were normalized using β-actin as a loading control. The relative density of the blot bands was quantified by densitometry using the ImageJ software.

Liver tissues were lysed with radio immunoprecipitation assay buffer (sc-24948; Santa Cruz Biotechnology Inc., Dallas, TX, USA) containing 1% protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO, USA) and 2% phenylmethanesulfonyl fluoride (P7626, Sigma-Aldrich). After determining the protein concentration, 40 μg of protein was separated by 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (0.45 μm; GE Healthcare Life Sciences, Marlborough, MA, USA). After blocking with 5% bovine serum albumin at 4°C for 4 h, the membranes were incubated with primary antibodies at 4°C for 12 h and subsequently incubated with the appropriate secondary antibodies at 4°C for 4 h (Table S1). The bands were then visualized using an enhanced chemiluminescence kit (PE0010; Solarbio Science & Technology Co., Ltd, Beijing, China) and a Tanon 5200 gel imaging system (Tanon Science & Technology Co., Ltd., Shanghai, China). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the bands.

Proteins from renal tissue were homogenized in ice-cold radioimmunoprecipitation assay lysis buffer (pH 7.0; sc-24948; Santa Cruz Biotechnology, Santa Cruz, CA, USA) supplemented with freshly prepared Halt Protease and Phosphatase Inhibitor Cocktail (78446; Thermo Scientific, Waltham, MA, USA). Tissue homogenate was centrifuged at 15,000× g for 15 min at 4 °C. The supernatant was separated for protein quantification, and a total of 30 μg protein was used for detection. Proteins were resolved via SDS-PAGE under reducing conditions unless otherwise noted. After gel electrophoresis, the proteins were electrotransferred from the gel onto polyvinylidene difluoride (PVDF) membranes (0.45-μm PVDF Transfer Membrane; Thermo Scientific). Then, the membrane was blocked with Rapid Block TM solution (VWR Life Science, Radnor, PA, USA) and probed with the indicated antibody. Images were obtained using the Syngene Western Blot Imager G: BOX (Syngene, Cambridge, UK) and analyzed by ImageJ software (NIH, Bethesda, MD, USA).

Kidney protein lysates were produced by homogenization in lysis buffer containing 50 mM Tris-HCl pH7.4, 0.5%NP40, 150 mM NaCl, 20 mM MgCl₂, protease and phosphatase inhibitors (Sigma Aldrich; 4906837001/5892970001), or in supplemented RIPA buffer (Santa Cruz; sc-24948). 1–25 μg protein lysate was reduced by addition of SDS-containing reducing buffer containing 0.1 M DTT and denatured by boiling at 95 °C for 5 min and loaded onto 4–12% Tris-HCl gels (ThermoFisher; NP0323BOX). Proteins were transferred to a nitrocellulose membrane, blocked with 5% BSA or 5% non-fat milk and probed with indicated antibodies. Antibodies; anti-NDRG1 D6C2 mAb (Cell Signaling Technologies; #9408, 1:1000) and anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology; #7074). Monoclonal anti-β-actin-peroxidase (Sigma; A3854, 1:10000) was used to detect the loading control and ECL prime (Amersham; RPN2232) was used for signal detection.