Goat anti rat igg h l hrp

Mineralocorticoid receptors were separated and identified by western blotting using commercial kits (Bio-Rad laboratories, USA). Briefly, proteins in kidney homogenate were quantified and separated by electrophoresis and blotted onto a nitrocellulose membrane. The membrane was incubated with rat anti-mineralocorticoid antibody (ab2774) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies; goat anti-rat IgG H&L (HRP) (ab205719) (Abcam, Laboratories Inc., USA) for 1 h at room temperature and 25 rev/min. Bound antibodies were detected by chemiluminiscence using Clarity Western enhanced chemiluminiscence (ECL) substrate (170–5060) and imaging was done using ChemiDoc Touch Imaging System (Bio-Rad laboratories, USA). Analysis of images was performed using Image Lab software (Bio-Rad laboratories, USA) and bands for mineralocorticoid receptors were normalized using housekeeping proteins (ß-actin, ab8227).

The expression of FLAG-tagged Rad52 was analyzed by western blotting. Proteins were extracted as described previously (32 (link)). Twenty micrograms of proteins (2 μg/μl) were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Any kD Mini-PROTEAN TGX Precast Gel (Bio-Rad). Transfer to membrane was performed with iBind Western System (Thermo Fisher Scientific) according to the manufacturer's protocol. Primary and secondary antibodies to detect Rad52-FLAG were FLAG M2 mouse monoclonal antibody (1:1000, Sigma-Aldrich) and goat anti-mouse IgG-HRP (1:2000, Santa Cruz Biotechnology), respectively. Primary and secondary antibodies to detect α-tubulin (loading control) were anti-alpha Tubulin antibody [YOL1/34] (1:2000, GeneTex) and goat Anti-Rat IgG H&L (HRP) (1:2000, Abcam), respectively. Following incubation with Clarity Western ECL Substrate (Bio-Rad), chemiluminescent signals were detected with ChemiDocTouch system (Bio-Rad). Gel images were processed with ImageJ software. The process involved cropping and altering window-level settings.

Imiquimod cream was purchased from Sichuan Mingxin Pharmaceutical Co., Ltd. (Sichuan, China). Atorvastatin was purchased from Pfizer Pharmaceutical Co., Ltd. (Liaoning, China). Hematoxylin and eosin (H&E) for HE staining were purchased from Leica (Wetzlar, Germany). OpalTM 4-Color Manual IHC Kit and Opal 620 Fluorophore were purchased from PerkinElmer (Waltham, MA, United States). Oxidized low-density lipoprotein (oxLDL) kit was purchased from Nanjing Jiangcheng Bioengineering Institute (Jiangsu, China). Antibodies for PCNA, PI3Kp110α, Akt, Phospho-Akt (Thr308), Phospho-Akt (Ser473), and Phospho-mTOR (Ser2448) were purchased from Cell Signaling Technology (Danvers, MA, United States). Antibodies for GAPDH, Ki67, loricrin, LOX-1, CD3, CD4, CD11c, F4/80, PI3Kp85α, mTOR, Phospho-mTOR (S2481), Goat Anti-Rat IgG H&L (HRP), and Rabbit Anti-Armenian hamster IgG H&L (HRP) were purchased from Abcam (Cambridge, UK). PierceTM bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Scientific (Cleveland, OH, United States). TRIzol reagent and all primers were purchased from Invitrogen (Waltham, MA, United States). PrimeScript™ RT reagent kit with gDNA Eraser and TB Green® Premix Ex Taq™ II (Tli RNaseH Plus), ROX plus, were purchased from Takara BioMed Co., Ltd. (Liaoning, China). ECL Ultra Western HRP Kit was purchased from Merck Millipore (Darmstadt, Germany).