Anti hsp27

The following antibodies were used in this study: anti-IGF-IIR (sc-25462, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CHIP (sc-66830, Santa Cruz Biotechnology), anti-HSF1 (sc-9144, Santa Cruz Biotechnology), anti-caspase-3 (sc-7148, Santa Cruz Biotechnology), anti-p53 (sc-126, Santa Cruz Biotechnology), anti-p-p53 (Ser15, sc-101762, Santa Cruz Biotechnology), anti-HSP27 (sc-1049, Santa Cruz Biotechnology), anti-HSP70 (sc-32239, Santa Cruz Biotechnology), anti-GAPDH (sc-47724, Santa Cruz Biotechnology), anti-HA (sc-7392, Santa Cruz Biotechnology), anti-Flag (sc-807, Santa Cruz Biotechnology), anti-β-actin (sc-8432, Santa Cruz Biotechnology), and anti-ubiquitin (sc-8017, Santa Cruz Biotechnology); anti-Flag (#ab1162, Abcam, Cambridge, UK); and anti-PARP (#9532, Cell Signaling Technology, Danvers, MA, USA), anti-caspase-3 (#9662, Cell Signaling Technology) and anti-FLT1 (#2893, Cell Signaling Technology). All secondary antibodies (anti-rabbit, mouse and goat, HRP-conjugated antibodies) were purchased from Santa Cruz Biotechnology. All reagents were purchased from Sigma.

We studied the cortex and the medulla separately to understand the effect of AG in both kidney regions. Western blot was realized as was previously published with some modifications [18 (link)]. Briefly, the renal cortex and the medulla were dissected and homogenized with an Ultra-Turrax homogenizer (Model PRO-200, PRO Scientific, Vernon Hills, IL, USA) in ice-cooled 10 mM Tris·HCl buffer at pH 7.4, supplemented with 1 mM EDTA, 1 mM EGTA, 0.25 M sucrose, 1% vol/vol Triton X-100, and a protease inhibitor cocktail (Complete Mini, Roche Applied Science # 5892970001). Tissue homogenates were subject to centrifugation. Step one was 900× g by 10 min. Next, the tissue was sonicated for 30 s on ice, vortexed, and centrifuged again by 2400× g and 17,000× g by 5 min. All procedures were held at 4 °C. Total proteins in supernatants were measured using the BCA Protein Assay Kit (ThermoFisher Scientific, Rockford, IL, USA), and samples were stored at −80 °C. The antibodies were: anti-HSP27 (sc-13132; Santa Cruz, OR, USA) and mouse anti-β-actin (A5441; Sigma, St. Louis, MO, USA) antibodies. Secondary antibodies were anti-mouse (A-21257) or anti-rabbit (A-21039) IgG conjugated with Alexa Fluor-750 (Thermo Scientific, South San Francisco, CA, USA). The resulting band intensities were quantified using Odyssey equipment and Image Studio Lite software (version 5.25; Li-Cor, Lincoln, NE, USA).

For protein extraction, 1 × 10⁶ cells from each condition were resuspended in lysis buffer (25 mM Tris-HCl, pH = 7.5; 150 mM NaCl; 1% NP-40; and 1 mM EDTA, pH = 8.0). Fresh 1 mM PMSF, 1 mM Na₃VO₄, and 1X Protease Inhibitor Cocktail-P2714 (Sigma–Aldrich) were added to the lysis buffer. The samples were vortexed for 5 min, followed by centrifugation at 13,000 × g at 4°C. The supernatant was collected, and the protein concentration was estimated using the standard Bradford method. The samples were then separated via SDS–PAGE and transferred onto a nitrocellulose membrane. The membrane was subsequently probed with primary antibodies including anti-HSP70 (SC-66048), anti-HSP27 (SC-13132), and anti-beta actin (SC-47778), followed by a secondary antibody, mouse anti-rabbit IgG-HRP (SC-2357) (Santa Cruz Biotechnology). The desired proteins were visualized using advanced ECL reagents (RPN2134, Cytiva). The density of each band was measured using Gel Analyzer version 19.01.