Anti srebp 1

Immunoblotting was performed as described previously [34 (link)]. Aliquots of nuclear extract (25 μg) and total lysate (50 μg) proteins extracted from livers were loaded onto 10% SDS-PAGE gels and transferred to PDVF membranes (Millipore, Darmstadt, Germany). The membranes were probed with anti-SREBP-1 (Santa Cruz Biotechnology, Dallas, USA), lamin A/C, phospho-JNK, and total JNK (Cell Signaling Technology, Denvers, USA) followed by horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit IgG (Cell Signaling Technology, Denvers, USA). Immune complexes were visualized using enhanced chemiluminescence (GE Healthcare Japan, Tokyo, Japan).

For immunoblotting analysis of the different proteins assessed in this study, cells were grown to 70–80% confluence and subjected to treatment. For total lysates’ preparation, cells were harvested and lysed in 200 µL of lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS) containing a mixture of protease inhibitors (aprotinin, phenylmethylsulfonyl fluoride, and sodium orthovanadate; Sigma). The same amounts of proteins from the total lysate or cytosolic fraction were resolved on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and probed with appropriate primary antibodies (Anti-SREBP1, Anti-SREBP2, Anti-GPAT, Anti-FASN, Anti-HMGR, Anti-LDLR, Santa Cruz Biotechnology, Santa Cruz, CA, USA and Merck KGaA, Darmstadt, Germany). To confirm equal loading and transfer, membranes were stripped and incubated with anti-GAPDH antibody [75 (link)] (Santa Cruz Biotechnology). The antigen–antibody complex was detected by incubating membranes with peroxidase-coupled goat anti-mouse antibody (Santa Cruz Biotechnology) and revealed using an ECL System (Bio-Rad Laboratories, Hercules, CA, USA) [76 (link),77 (link)]. Blots were then exposed to film, and the bands of interest were quantified using ImageJ software (version 1.52a) [78 (link)].

ChREBP and SREBP-1 protein expression in maternal and fetal livers was assessed by western blotting. Liver tissues (100 mg) were homogenized as described above. Proteins (40 μg/lane, determined by BioRad protein assay) were separated by electrophoresis using Nu-PAGE® gels (Life Technologies, Grand Island, NY) and transferred to PVDF membranes (Millipore). The blots were probed with anti-SREBP-1 (Santa Cruz Biotechnology, Dallas TX), anti-ChREBP (Novus Biologicals, Littleton, CO), and anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) antibodies, followed by near infrared-fluorescently labeled secondary antibodies (LI-COR, diluted 1:15,000), and revealed using the Odyssey infrared imaging system (LI-COR Biosciences), as previously described [28 (link), 32 (link)]. Band densities (SREBP-1: GAPDH and ChREBP:GAPDH) were assessed using Image J software (NIH).

After de-paraffinization, the sections were incubated with a 3% H₂O₂ solution to block endogenous peroxidases. Antigen retrieval was carried out using 0.1 M sodium citrate (pH 6.0) for 60 min. Sections were incubated with anti-α-SMA (1:100; BIOSS), anti-desmin (1:100; Abcam), anti-TGF-β1 (1:100; Santa Cruz Biotechnology, Inc.), anti-NF-κB p65 (1:100; Affinity Biosciences) or anti-SREBP-1 (1:100; Santa Cruz Biotechnology, Inc.) antibodies overnight at 4 °C, and a horseradish peroxidase-conjugated secondary antibody and diaminobenzidine substrate were added sequentially. Following hematoxylin counterstaining and dehydration, the sections were mounted and observed under a Leica DM4000B photomicroscope (Leica Microsystems, Inc.).

Proteins were extracted from the cultured cells, supernatants and the xenograft tumours using Complete Lysis-M buffer (Roche). Equal amounts of protein were incubated with anti-FASN (BD Bioscience), anti-SREBP-1 (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-AKT, anti-phospho-AKT (P-AKT, Ser⁴⁷³), anti-ERK1/2, anti-phospho-ERK1/2 (P-ERK1/2, Thr202/Tyr204), anti-AMPK, anti-phospho-AMPK (P-AMPK, Thr172) or anti-beta-actin (Cell Signaling Technology) antibody.

Total protein samples were extracted from vehicle- or DFE (20 µg/mL)-treated PCa cells by PRO-PREP Protein Extraction Solution (iNtRON technology, Seoul, South Korea) adding with protease inhibitors. A Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific) was utilized to determine the concentrations of proteins. The equal amounts of protein samples were loaded into SDS-PAGE gels for electrophoresis and then proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the blotted membranes were blocked using 5% non-fat milk in PBS with Tween-20. After blocking, the membranes were incubated with primary antibodies for overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Primary antibodies were used in this study as follow: anti-AR, anti-SREBP-1, anti-FASN (Santa Cruz Biotechnology, Dallas, TX, USA), anti-SREBP-2 (abcam, Cambridge, MA, USA), anti-caspase-3 (Novus Biologicals, Littleton, CO, USA), anti-PARP (GeneTex, Irvine, CA, USA), and anti-β-actin (Millipore, Burlington, MA, USA). The protein signals were visualized using an Enhanced Chemiluminescence Kit (Amersham Biosciences, Arlington Heights, IL, USA) and an imaging system (ImageQuant LAS 4000; GE Healthcare, Pittsburgh, PA, USA). The ImageJ software was utilized to quantify specific protein bands by normalized a loading control, β-actin.