Supersignal enhanced chemoluminescent substrate

Lysates from the cells and microdissected renal tubules from each experimental group were separated in parallel on two 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% nonfat milk in 0.1% tris buffered saline with Tween-20 (TBST), and probed using antibodies to rabbit polyclonal anti-SNAIL antibody (1:100, ab180714, Abcam, Cambridge, UK), mouse anti-E-CADHERIN antibody antibody (1:100, ab76055, Abcam, Cambridge, UK), mouse monoclonal VIMENTIN (D21H3) antibody (1:100, #5741, cell signaling technology, MA, USA), and rabbit polyclonal anti-COLLAGEN IV antibody (1:100, ab6586, Abcam, Cambridge, UK) at 4 °C overnight. After washing, the secondary antibody (horseradish peroxidase-labeled IgG anti-rabbit/mouse antibody, Invitrogen, USA) was used at 1:3000 dilution for 1 hour at room temperature. The supersignal-enhanced chemoluminescent substrate (Pierce Biotechnology, Inc., Rockford, IL, USA) was applied to the probed membrane and exposed for 10 minutes before the protein bands were visualized on radiograph films (Super Rx, Fuji Photo Film, Tokyo, Japan).

Lysates from the cells and microdissected renal tubules from each experimental group were separated in parallel on two 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% nonfat milk in 0.1% tris buffered saline with Tween-20 (TBST), and probed using antibodies at 4°C overnight. Primary antibodies against PPARγ (1:100, ab19481), TGF-β1 (1:100, ab27969), total Smad3 (1:100, ab40854), Smad3 (phospho S213) (1:100, ab63403), CTGF (1:100, ab6992), Fibronectin (1:200, ab2413), Collagen I (1:200, ab6308) and beta Actin (1:200, ab6276) were purchased from Abcam (Cambridge, USA). After extensive washing in TBST buffer, the secondary antibody (horseradish peroxidase-labeled IgG anti-rabbit/mouse antibody, Invitrogen, Cambridge, MA) was used at 1:3000 dilution for 1 hour at room temperature. The supersignal-enhanced chemoluminescent substrate (Pierce Biotechnology, Inc., Rockford, IL) was applied to the probed membrane and exposed for 10 minutes before the protein bands were visualized on radiograph films (Super Rx, Fuji Photo Film, Tokyo). Quantification was performed by measurement of the intensity of the bands using ImageJ analysis software (National Institutes of Health, Bethesda, MD).