Image lab version 5

Western blot was performed as previously described (23 (link), 24 (link)). Briefly, 50 mg of spleen tissue was homogenized with protein extraction solution (iNtRon biotechnology, Sungnam, Korea) according to the manufacturer's instructions. After determining protein concentrations, 50 μg samples were boiled and subjected to 12% SDS-PAGE for each lane. After transferring and blocking to a polyvinylidene difluoride (PVDF) membrane, the membrane was incubated with anti-SIRT1 (Abcam, Cambridge, MA, USA) at 1:500 for over-night. After washing and incubation for 2 h with peroxidase-labeled secondary antibody diluted 1:1,000 in T-BST, the blot image was obtained using a Bio-Rad Molecular Imager ChemiDocMP and quantified by Image Lab version 5.0 software (Bio-Rad, Richmond, CA, USA).

Western blot was performed as previously described [60 (link)]. Each 50 mg of spleen tissue was homogenized with protein extraction solution (iNtRon biotechnology, Sungnam, Korea) according to the manufacturer’s instructions. After protein concentrations were determined, 50-µg samples were boiled and subjected to 12% SDS-PAGE for each lane. After transferring and blocking to a polyvinylidene difluoride (PVDF) membrane, the membrane was incubated for 2 h with anti-histone H3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or H4 antibody (Santa Cruz) at 1:500. After washing and incubation for 2 h with peroxidase-labeled secondary antibody diluted 1:1000 in T-BST, The signal was obtained using a Bio-Rad Molecular Imager ChemiDocMP and quantified by Image Lab version 5.0 software (Bio-Rad, Richmond, CA, USA).

After treatment for 24 h as described, the cells were collected and lysed with RIPA buffer (Tris-HCl: 50 mM (pH 8.0); NP-40 : 1.0%; Na-deoxycholate: 1.0%; NaCl: 150 mM; SDS: 0.1%; and PMSF: 0.05 mM), and the protein concentration was assessed using the BCA Protein Assay Kit (Abcam, Cambridge, UK). Equal amounts of protein lysates mixed evenly with 6× loading buffer (5 : 1, V/V) were transferred to PVDF membranes. After blocking in 5% BSA for 2 h, the membranes were incubated with antibodies against p21 and p27 (1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA), SM22ɑ, (1 : 1000, Abcam, Cambridge, UK), and mouse anti-rat/rab β-actin (1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. Then, the membranes were incubated with HRP-conjugated secondary antibody at room temperature for 1 to 2 h, and chemiluminescent autography was performed using an ECL kit (Beyotime Biotechnology, Beijing, China). The grey values of the protein bands were analysed using the image processing software Image-Lab version 5.0 (Bio-Rad; Hercules, CA, USA).

Total RNA was isolated from the rat tissues using TRI-Reagent (Molecular Research Center, Inc. Cincinnati, USA) according to the manufacturer’s protocol. Northern blot analysis was performed as described previously [11 (link)]. Briefly, total RNA (20 μg) was separated by electrophoresis through a 1.2% (w/v) agarose gel containing 6.5% (v/v) formaldehyde. After blotting on a sheet of Bio-Rad Zeta-Probe GT Blotting Membrane (Bio-Rad Laboratories, Richmond, CA, USA), RNA samples were hybridized with [α-³²P] dCTP-labeled cDNA probes, followed by washing under stringent conditions. Each blotted membrane was placed on a sheet of Fuji Medical X-ray film (Fujifilm Co., Tokyo, Japan) with an intensifying screen at −80°C. Target and 18S ribosomal RNA bands were visualized and quantified using an image scanner (ChemiDoc XRS Plus Imaging System, Bio-Rad, USA) and image analysis software (Image Lab Version 5.0, Bio-Rad). The relative amounts of hybridized radiolabeled cDNAs were normalized to 18S ribosomal RNA levels to correct for differences in sample loading.

Total RNA was isolated from the kidney tissues using TRI Reagent® (Molecular Research Center, Inc. Cincinnati, USA) according to the guidelines provided by the manufacturer. Northern blot analysis was carried out as previously reported [12 (link)]. Briefly, electrophoresis on a 1.2% (w/v) agarose gel with 6.5% (v/v) formaldehyde was used to separate total RNA (20 μg). After blotting on a Bio-Rad Zeta-Probe GT Blotting Membrane (Bio-Rad Laboratories, Richmond, CA, USA), the RNA was hybridized with [α-32P] dCTP (PerkinElmer, Inc., Japan)-labeled complementary DNA (cDNA) probe against HO-1 (provided by Dr. S Shibahara, Sendai University, Sendai, Japan) and ALAS1 (provided by Dr. M Yamamoto, Tohoku University, Sendai, Japan), followed by washing under appropriate conditions. Each blotted membrane was exposed to a sheet of Fuji Medical X-ray film (Fujifilm Co., Tokyo, Japan) with an intensifying screen at −80°C. Signals corresponding to the target mRNA on the film and 18S ribosomal RNA bands visualized on the gel were estimated using an image scanner (ChemiDoc XRS Plus Image Processing System, Bio-Rad, USA) and image analysis software (Image Lab™ version 5.0; Bio-Rad Laboratories). The relative amounts of hybridized radioisotope-identified cDNAs were normalized to 18S ribosomal RNA levels to correct for sample loading differences.