Secondary anti rabbit

For immunoblotting, primary antibodies against androgen receptor (#D6F11XP^®), mTOR (#2983), S6K1 (#2708), eEF2 (#2332), AMPK (#2532), ULK1 (#6439), p62 (#8025), LC3B (#2775), Beclin (#3495) and BNIP3 (#44060), were purchased from Cell Signaling Technology (Beverly, USA). Primary antibodies against 5α-reductase 2 (#293232), MuRF-1 (#sc-398608), UBR5 (#sc-515494), TOM20 (sc-136211) and RPS6 (#sc-74459), were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Primary antibodies against MAFbx (#92281), Porin (#154856), OPA1 (#157457), DRP1 (#184247), FIS1 (#156865), MFN2 (#56889) and Mitofilin (#137057), were purchased from Abcam (Cambridge, UK). All antibodies were diluted 1:1000 except for 5α-reductase, MuRF-1, UBR5, S6, Beclin, LC3B, OPA1, MFN2 which were diluted 1:500, and eEF2 and Porin which were diluted 1:2000. Secondary anti-mouse (#7076, 1:10000) and secondary anti-rabbit (#7074, 1:10000) were purchased from Cell Signaling Technology.

Approximately 100 mg of frozen kidney was taken and pulverized to a fine powder, suspended in 900 µl of T-PER (Pierce, Rockford, IL, USA) containing 1 % protease and phosphatase inhibitors and homogenized. Supernants were collected by centrifugation (10,000×g for 10 min at 4 °C and the protein concentration determined using the 660 nm assay (Pierce, Rockford, IL, USA). Samples were equally diluted with 4× Laemilli buffer and then subjected to electrophoresis on 10 % PAGEr Gold Precast gel (Lonza, Rockland, ME, USA) before transfer to nitrocellulose membranes as detailed elsewhere [44 (link)]. Equal loading of protein was verified by Ponceau S staining of nitrocellulose membranes. Membranes were blocked with 5 % milk in TBST for 1 h and later probed with primary antibodies against p-stat-3 (Tyr 705), stat-3, cleaved caspase-3 and caspase-3 (Cell Signaling Technology, Danvers, MA, USA). After exposure to the primary antibodies, membranes were washed with TBST (3 × 5 min) and incubated with secondary anti-rabbit (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Immunoreactivity was visualized using Supersignal West Pico Chemiluminiscent substrate (Pierce, Rockford, IL, USA) and quantified by Fluorchem 9900 software (Protein Simple, Santa Clara, CA, USA).

Western blot analyses were performed as previously described [18 (link),21 (link)]. Samples were resolved on denaturing 10% polyacrylamide SDS gels followed by transfer to nitrocellulose membranes. Membranes were blocked for 1 h in PBS (5% non-fat dry milk; 0.1% Tween-20) and incubated overnight with antibodies anti-Ym1 (#60130, 1:1000—StemCell Technologies, Vancouver, BC, Canada), anti-P-STAT3 (#9134, 1:1000–Cell Signaling Technology, Beverly, MA, USA) or anti–β-actin (1:10,000, #A5316–AC-74, Sigma-Aldrich, St. Louis, MO, USA). Secondary anti-rabbit (#7074, Cell Signaling Technology) or anti-mouse (#sc-2005, Santa Cruz Biotechnology, Dallas, TX, USA) HRP-conjugated antibodies (1:3000) were added to the membranes and incubated for 1 h at room temperature. ECL detection system (GE Healthcare, Chicago, IL, USA) was used to visualize immunoreactive bands. Autoradiographs were scanned, and densitometry analysis was conducted in ImageJ software (Bethesda, MA, USA). Results were expressed as arbitrary units and normalized using β-actin as loading controls.

Cell samples underwent lysis with cell lysis buffer supplemented with protease inhibitors (Beyotime) on ice for 20 min. Protein quantitation applied the BCA protein assay kit (Beyotime). Equal amounts of total protein were resolved by 10% SDS-PAGE, followed by electro-transfer onto polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Next, a 1-h blocking was performed with 5% nonfat milk, followed by successive incubations with anti-TLR4 primary (Cell Signaling Technology, USA; Cat^#: 38519, 1:1000) and anti-rabbit secondary (Cell Signaling Technology, Cat^#: 5151, 1:30000) antibodies at 4°C (overnight) and ambient (1 h), respectively. An Odyssey infrared imaging system (LI-COR Biotechnology, USA) was utilized for data acquisition. Quantitation utilized ImageJ (National Institutes of Health, USA), with β-actin for normalization.