Ecl western blotting detection reagent

Cells were lysed in Laemmli buffer (62.5 mM TrisHCl, 2% SDS, 10% Glycerol, 0.1% Bromphenolblue, 5 mM β-mercaptoethanol). Whole-cell lysates were separated on SDS polyacrylamide gels and electroblotted onto nitrocellulose membranes (Whatman, Maidstone, UK). Membranes were blocked for 1 h in 5% (w/v) nonfat milk in TBS-T (20 mM Tris base, 137 mM sodium chloride, 0.1% Tween 20, pH 7.6). After blocking, the membranes were probed with antibodies against histone 3 (H3, dilution 1:5000, #ab1791, Abcam, Cambridge, UK), acetyl-H3 (acH3, dilution 1:500, #06-599, Millipore, Burlington, MA, USA), H3K27ac (dilution 1:600, #ab4729, Abcam), H3K18ac (dilution 1:1000, #39588, active motif), or α-tubulin (dilution 1:10,000, ab7291, Abcam). As secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit (dilution 1:5000, #111-036-047, Jackson ImmunoResearch, Philadelphia, PA, USA) or goat anti-mouse antibodies (dilution 1:5000, #115-036-062, Jackson ImmunoResearch) were used. Signals were detected using the ECL Western blotting detection reagents (Advansta, San Jose, CA, USA) and the Fusion FX software (Vilber, Sursee, Switzerland).

Protein extraction from the kidney cortex or cultured podocytes under different experimental conditions was conducted as previously described [22 (link)]. The nuclear protein is isolated and prepared as described in the Nuclear and Cytoplasmic Protein Extraction Kit (Nanjing KeyGEN Biotech, Nanjing, China). According to the manufacturer’s protocol, the protein concentration was evaluated using a protein assay reagent kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Equal amount of proteins was separated on 9% sodium dodecyl sulfate–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% non-fat dry milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-ATF3 (Abcam, Cambridge, MA), rabbit anti-NFATc1(Abcam), rabbit anti-Histone (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Bax (Santa Cruz, Dallas, TX, USA), rabbit anti-Bcl-2 (Cell Signaling Technology), rabbit anti-GAPDH (Bioworld Technology, Nanjing, China), and rabbit anti-Histone (Cell Signaling Technology). Finally, membranes were detected using ECL Western Blotting Detection Reagents (Advansta, Menio Park, CA, USA).

Western blot analysis was performed using antibodies that specifically recognize proteins, including Glut4 and glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The epididymal adipose tissue was homogenized, proteins were extracted, and 15 μg of extracted protein was loaded for sodium dodecyl sulfate–polyacrylamide gel electrophoresis immunoblot analysis. Protein bands were then transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking the nonspecific sites, the membrane was probed with primary antibodies, followed by a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Inc., Danvers, MA, USA). Detection of antibody reactions was performed with ECL Western blotting Detection Reagents (Advansta, San Jose, CA, USA). Each band was normalized using the corresponding value of Gapdh as an internal control. The antibodies used were Gapdh (primary antibody (mouse monoclonal, Abcam, Cambridge, UK) 1:4000 dilution, secondary antibody (rabbit polyclonal, Abcam) 1:4000 dilution) and Glut4 (primary antibody (rabbit monoclonal, Cell signaling) 1:1000 dilution, secondary antibody (rabbit polyclonal, Abcam) 1:4000 dilution). Reaction times were overnight at 4 °C for primary antibodies and 1 h at room temperature for secondary antibodies.