Ab19481

After treatment with stearate, oleate or linoleate, cells were collected and total proteins were lysed using RIPA lysis buffer (Beyotime, Nanjing, China). The homogenates were combined with equal volumes of SDS sample buffer, and the proteins were separated by electrophoresis on a 5~12% polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20, followed by overnight probing with the following primary antibodies: (1) CD36 (N-15) antibody (sc-5522, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (2) ACACA (T-18) antibody (sc-26817, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (3) DGAT1 antibody (ab59034, 1:500, Abcam, Cambridge, MA, USA), (4) SREBP1 (C-20) (sc-366, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (5) PPARγ (T-18) antibody (ab19481, 1:500, Abcam, Cambridge, MA, USA), (6) β-actin (C4) antibody (sc-47778, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). β-actin was intended to serve as a loading (internal) control. After washing, membranes were incubated with secondary antibody (ABR, Golden, CO, USA) and conjugated to HRP. The chemiluminescent signal was detected using ECL reagents (Beyotime, Nanjing, China) and bands were quantified by image processing software (Image Pro Plus 6.0, Media Cybernetics, Rockville, MD, USA).

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).