Gapdh sc 20357

After transfection for 48 h, glioma cells were harvested and lysed with the NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris/HCl (pH 8.0), 1 mM EDTA) on ice for 30 min in the presence of protease inhibitor. The protein concentration was quantitated with the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Twenty micrograms protein of each sample was loaded and separated by the SDS/PAGE (15% gel). The protein was then transferred on to the nitrocellulose membrane (Millipore, Billerica, MA, U.S.A.) and blocked with 5% of non-fat milk at RT for 1 h. Subsequently, membrane was incubated with primary antibodies against PTEN (ab32199, 1:2000 dilution, Abcam), GAPDH (sc-20357, 1:3000 dilution, Santa Cruz Biotechnology, Inc., Dallas, TX, U.S.A.), SP1 (#5931, 1:2000 dilution, Cell Signaling Technology, Danvers, MA, U.S.A.) for 2 h at RT, respectively. After washing with TBS and Tween-20 (TBST), the membrane was incubated with the secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h at RT. The protein bands were detected with ECL (Amersham Pharmacia Biotech, Little Chalfont, U.K.) and analyzed using ImageJ Software (version 1.62; National Institute of Health, Bethesda, MD, U.S.A.).

For Western blot analyses, deep-frozen kidney samples were homogenized in a Micro-Dismembrator S (Sartorius Stedim Biotech GmbH, Göttingen, Germany) at 3000 rpm for 30 s and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X100, 2 mM EDTA, 2 mM EGTA, 40 mM β-glycerophosphate, 50 mM sodium fluoride). Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membrane, and utilized in Western blot analysis using antibodies as indicated. Antibody against FN1 (ab2413) was obtained from Abcam (Cambridge, UK). The GAPDH (sc-20357) and CTGF (sc-14939) antibodies came from Santa Cruz Biotechnology (Heidelberg, Germany). HAVCR/KIM-1 (#AF1817) and Lipocalin-2 (#AF1857) antibodies were from R&D Systems (Minneapolis, MN, USA). Secondary antibody donkey anti-goat IgG, HRP conjugate (#AP180P) was obtained from EMD Millipore (California, CA, USA) and secondary antibody donkey anti-rabbit IgG, HRP conjugate (#NA934) was obtained from Sigma-Aldrich (Munich, Germany).

For Western blot analyses, 8 μg of protein were loaded in each lane. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, El Prat de Llobregat, Spain) overnight at 4°C and 100 mA. Reversible Ponceau staining was used as a loading control (39 (link)). The PVDF membranes were blocked with 5% skimmed milk powder in Tris-Buffered Saline and Tween 20 (TBS-T) for 1 h 15 min and probed with rabbit polyclonal Pparγ (sc-7196) and goat polyclonal Gapdh (sc-20357) primary antibodies (Santa Cruz Biotechnology, CA, USA) at a dilution of 1:200 overnight at 4°C on a tube rotator. Membranes were washed in TBS-T and probed with a horseradish peroxidase-conjugated anti-rabbit (sc-2004) or anti-goat (sc-2020) secondary antibody (Santa Cruz Biotechnology, CA, USA) at a dilution of 1:10,000 for 1 h 15 min. Membranes were washed with TBS-T and chemiluminescent detection performed using an enhanced chemiluminescence kit (Pierce ECL Western blotting Substrate; Thermo Scientific, Alcobendas, Spain). Western blotting results were obtained from two independent cultures, and band intensities were quantified by scanning densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to Ponceau staining.