Hrp linked goat anti rabbit igg

Kidney tissue and whole cell extracts were incubated in lysis buffer (150 mmol/l NaCl, 50 mmol/l Tris-Cl, 1 mmol/l EDTA, 1 mmol/l PMSF, 1 mmol/l Na₃VO₄, 1 mmol/l NaF, 1% NP-40, 0.25% deoxycholic acid and 1 μg/ml leupeptin) for 20 min on ice, followed by 15 min centrifugation at 1500 rpm. Extracts in loading buffer were electrophoresed on 8~12% acrylamide gels and the proteins were transferred onto PVDF membranes in transfer buffer for 1 h at 120V. Membranes were blocked for 1 h in TBST [20 mmol/l Tris-HCl (pH 7.6); 137 mmol/l NaCl; and 0.1% Tween 20] with 5% skim milk, and then incubated overnight at 4°C with one of the following antibodies: His-probe (H-15), MT (N-19), SOD-1, NF-κBp65 (F-6), total LRP6, total GSK-3β (H-76), phospho-GSK-3β (Ser 9), β-catenin (H-102), VEGF (A-20), CTGF (H-55), α-tubulin and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-p38, phospho-ERK, phospho-JNK, phospho-AMPKα (Thr172), phospho-LRP6 (Cell Signaling Technology, Beverly, MA); fibronectin (Sigma, St. Louis, MO); RAGE from Abcam (Cambridge, MA). Membranes were thoroughly washed with TBST and then incubated with horseradish peroxidase conjugated secondary antibody at 1:20000 dilution (HRP-linked goat anti-rabbit IgG; Santa Cruz Biotechnology) washed in TBST and detected with an ECL system (iNtRON Biotechnology, Ansan, Korea).

Proteins were extracted with ice‐cold RIPA buffer supplemented with protease inhibitor cocktail tablets (Roche Diagnostics GmbH) and 1 mmol/L phenylmethylsulphonyl fluoride. Then, lysates were incubated on ice for 30 minutes and centrifuged at 9600 g at 4°C for 15 minutes. Equal amounts of protein (30‐50 μg protein/lane) were separated by SDS‐PAGE gels and transferred onto PVDF membranes as previously described.21 The membranes were blocked with 5% non‐fat milk in TBST for 1 hour at room temperature and then incubated with primary antibodies against iNOS (1:1000; Abcam), Arg1 (1:1000; ProteinTech Group), NLRP3 (1:1000; Abcam) or Caspase1 (1:1000; Abcam) overnight at 4°C on a rocking platform. The β‐tubulin (1:5000; Santa Cruz) was used as an internal control. After washing with TBST three times for 10 minutes each, the membranes were further incubated with appropriate secondary antibodies (HRP‐linked goat anti‐rabbit IgG or HRP‐linked goat anti‐mouse IgG [1:4000; Santa Cruz]) for 2 hours at room temperature. After washing three more times, the immunoblots were developed by routine enzymatic chemiluminescence. The signalling was quantified by Image J software and presented as normalized to β‐tubulin.