Anti mouse or anti rabbit peroxidase

For each sample, 50 µg of renal tissue protein extract was used for electrophoresis on 10% polyacrylamide gels (SDS-PAGE). The immunostaining was carried out with primary antibodies (β-actin/1:1,000, Sigma, USA; IKK-α/1:1,000, Cell Signaling, USA; Argonaute 2/1:1,000, Cell Signaling, USA; Drosha/1:1,000, Cell Signaling, USA; Dicer/1:1,000, Imgenex, India), followed by conjugated secondary antibodies (anti-mouse or anti-rabbit peroxidase/1:5,000, Sigma, USA). Then, the membrane was revealed by chemiluminescence methods using the ECL kit (Millipore, USA), and the images were acquired on GEN-BOX equipment (Syngene, UK) (n = 5). The GeneSnap software and GeneTools (Syngene, UK) were used to identify, analyze, and quantify the gel bands.

For each kidney sample total protein was isolated using RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and global protein was measurement by BCA™ Protein Assay Kit (Pierce, EUA). Around 50 μg of tissue protein was utilized for SDS-PAGE electrophoresis on 12.5% polyacrylamide gels. The immunostaining was performed with primary antibodies (Anti-Cofilin/1:1,000, Abcam, USA, Anti-PPAR-α/1:300, Abcam, Anti-CPT1A/1:1,000, Abcam, Anti-CPT-2/1:500, Abcam and Anti-Beta Actin/1:5,000, Cell Signaling, USA), followed by conjugated secondary antibodies (anti-mouse or anti-rabbit peroxidase/1:250.000, Sigma, USA). After, the nitrocellulose membrane was revealed by chemiluminescence methods using the ECL kit (Millipore, USA), and the images were acquired on Amersham Imager 600 equipment (GE Healthcare, UK). The SyncMaster 740 n software device (GE Healthcare, UK) were used to analyze and quantify the gel bands. The experiment was reapeated twice.