Bm chemiluminescence kit

Western blot analysis of kidney tissue homogenates was performed to analyze SNAIL1, TWIST1, SMAD3, and SMAD7 protein expression (Invitrogen, Waltham, MA, USA). According to our previous real-time PCR analysis, GAPDH (Invitrogen, Waltham, MA, USA) was also used as a housekeeping protein. Proteins were extracted from kidney tissue using P-TER solution (Thermo Fisher Scientific, USA) and quantified by the BCA protein assay kit (Thermo Fisher Scientific, USA). A quantity of 50 µg of total proteins were separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA, USA). Blocking was performed using 5% non-fat dry milk in 1X TBST for 1h at 4 °C with constant agitation. Membranes were incubated overnight at 4 °C with primary antibodies diluted 1:1000 (SNAIL1, TWIST1, SMAD3, and GAPDH) and 1:500 (SMAD7) in 1X TBST. Antibody binding was revealed with an HRP-conjugated secondary anti-antibody diluted 1:5000 in 1X TBST using a BM Chemiluminescence kit (Roche Diagnostics, Indianapolis Ind, Indianapolis, IN, USA). Densitometric analysis was performed with a UVP ChemiStudio image analyzer (Analytik Jena, Jena, Germany) using the VisionWorks^® software (Analytik Jena, Germany). The semiquantitative analysis of every protein was shown as normalized levels.

To test for protein stability over time, cyclohexamide-treated X. laevis explants were boiled in sample buffer, microfuged briefly, and proteins were separated by SDS-PAGE before transferring onto a PVDF (polyvinylidene fluoride) membrane by electroblotting. Membranes were blocked in 5% Marvel milk powder/PBST (phosphate buffered saline with Triton-X 100) for 1 h before incubating with the primary antibody of anti-HA (Sigma) at a concentration of 1 in 4000 overnight at 4 °C. After extensive washing, the membrane was incubated in a secondary antibody (1 in 4000 goat anti-mouse conjugated with HRP (Invitrogen) for an hour at room temperature. Membranes were washed and visualised using a BM Chemiluminescence kit (Roche) and hyperfilm ECL. After stripping in 50 mM Tris-HCl (pH 7) with 2% sodium dodecyl sulfate (SDS) and 50 mM dithiothreitol (DTT), the antibody β-tubulin (Cell Signalling Technology), at a concentration of 1 in 10,000, followed by a goat anti-rabbit secondary (Invitrogen) at a concentration of 1 in 2000, provided a normalisation control using the same method.

Powdered liver tissue, was homogenized (10% w/v) in homogenization buffer containing 50 mM Tris-Hcl, 150 mM Nacl, 5mM Sodium Pyrophosphate (NaPPi), 50mM NaF, 1mM EDTA, 1mM dithiothreitol (DTT), 0.1%SDS (w/v), 1% TXT-100 (v/v), and protease inhibitor cocktail. Tissue homogenates were centrifuged at 1000g for 10 min at 4 °C and supernatants were stored in 50 µl aliquots at - 80 °C for further analysis. Bradford Protein Assay kit was used to evaluate the protein content of the supernatant. SDS-polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose membranes was performed using 50 μg of homogenate protein. Then membranes were blocked in 5% non-fat milk in Tris-buffered saline Tween-20 and incubated overnight with rabbit antibodies against phospho-AMPK (p-AMPKThr172) and AMPK (1:1000 dilution - Cell Signaling Technology Inc., Danvers, Massachusetts, USA) in 5 % BSA (wt/vol). After extensive washing, the membranes were incubated with a peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000 dilution, Cell Signaling Technology Inc. Danvers, Massachusetts, USA) in 5% skim milk (wt/vol). After washing, antibodies were visualized using the BM Chemiluminescence kit (Roche; Germany). Densitometric analyses of immunoblots were performed using Image J software (National Institute of Health, Bethesda, Maryland).^{26 (link)}