Goat anti rabbit igg

Protein extracts were made by TCA precipitation (Foiani et al. 1994 (link)) from 3 ml of culture. Gel electrophoresis, blotting, and probing were performed as described (Kaur et al. 2018 (link)). Primary antisera and dilutions: mouse anti-HA monoclonal (clone 12CA5, 11583816001; Roche), 1/10,000; rabbit anti-MYC (Santa Cruz Biotechnology, sc-789), 1/1000; goat anti-ARP7 (Santa Cruz Biotechnology, sc-8961), 1/1000. Secondary antibodies were alkaline phosphatase conjugates of goat anti-mouse IgG (A3562; Sigma); rabbit anti-goat IgG (A4187; Sigma); and goat anti-rabbit IgG (A3687; Sigma). All were used at 1/10,000 dilution.

Preparation of protein extracts from subconfluent cell cultures or resected tumors using RIPA buffer as well as western blot analysis was performed as described elsewhere [58 (link)]. In brief, 80 µg of proteins were separated in a 8.5% v/v sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Fisher Scientific, Schwerte, Germany) using a semi-dry transfer system (Bio-Rad, Munich, Germany). After blocking with 5% w/v skimmed milk for at least 1 h, PVDF membrane was incubated with goat anti-human EphB4 IgG (R&D, AF3038; 0.2 µg/mL) or rabbit anti EphrinB2 IgG (Novus Biologicals, Littleton, CO, USA, NBP1-48551; 3.1 µg/mL) for 2 h at room temperature and afterwards overnight at 4 °C. Membranes were washed three times and incubated with the appropriate horseradish peroxidase coupled secondary antibodies (rabbit anti-goat IgG, Sigma Aldrich, Taufkirchen, Germany; A5420; goat anti rabbit IgG, Sigma Aldrich, A0545) for 1 h at room temperature. Protein bands were detected with Super Signal West Pico, Dura, or Femto Chemiluminescent Substrate (Fisher Scientific) and the MF-ChemiBis 3.2 imaging system (Biostep, Burkhardtsdorf, Germany). To verify equal protein loading, membranes were stripped and reprobed with mouse anti β-actin IgG (Sigma Aldrich, A5316) and horseradish peroxidase coupled rabbit anti-mouse IgG (Sigma Aldrich, A9044).

Cell culture materials were obtained from Invitrogen (Burlington, Ontario, Canada). The reagents 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI) and the PureLink™ HiPure Plasmid DNA Purification Kit were purchased from Sigma (St. Louis, MO, USA). Primary antibodies against PARP and Twist were purchased from GeneTex (Beverly, MA, USA). Anti-HER-2, anti-phospho-Akt (Ser473), anti-E-cadherin, anti-vimentin, anti-Snail, and anti-Slug antibodies were purchased from Cell Signaling (Beverly, MA, USA). Primary antibodies against ILK, phosphor-ILK (Thr173), phospho-mTOR (Ser2448), and phospho-GSK3β (Ser9) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against YB-1 and β-Actin were purchased from Millipore (Temecula, CA, USA). The antibody against HIF-1α was purchased from BD Biosciences Clontech (San Jose, CA, USA). For Western blotting, the secondary antibodies of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Millipore (Temecula, CA, USA), and enhanced chemiluminescence (ECL) reagents were purchased from Sigma-Aldrich. Emodin, AE, and rhein were purchased from Sigma-Aldrich. The RNAi Consortium of YBX-1 was selected by the National RNAi Core Facility.

A lysis buffer [NaCl (140 mM), Tris–HCl (25 mM; pH 7.4), and 1% NP-40] and freshly prepared protease inhibitor cocktail were used for harvesting the liver tissue extracts from the experimental rats. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins was performed on a 10–15% agarose gel and transferred to PVDF membranes (BioRad, Hercules, CA). After blocking in 5% skim milk in PBS, the membrane was incubated with each primary antibody for SREBP1 and HMGCR (Abcam, Cambridge, MA, UK) and AMPK, p-AMPK, ACC, p-ACC and β-actin (Cell Signaling, Danvers, MA, USA), and then diluted 1:1000 with 1% skim milk in PBS at 4°C overnight. Blots were then incubated with peroxidase-conjugated secondary antibody goat anti-rabbit IgG (Millipore, Temecula, CA, USA) diluted 1:10,000 at room temperature for 1 h. Immunoreactive bands were detected by a Super Signal West Dura Extended Duration Substrate (Thermo, CA, USA), following the manufacturer's instructions. A Chemi Imager Analyzing System (Alpha Innotech, CA) was used for the densitometric analysis directly from the blotted membrane.

Twelve serum samples from PB or BMA were collected for Western blotting. The protein content was quantitated using a protein assay kit (Pierce Biotechnology, Rockford, IL, USA), separated by 6% SDS-PAGE for catalase and 10% for glutathione peroxidase and β-actin, and transferred onto membranes using a transfer unit (Bio-Rad, Hercules, CA, USA). After blocking, the membranes were incubated with 1,000-fold diluted rabbit antibodies against catalase and glutathione peroxidase (Abcam, Cambridge, UK) or mouse antibodies against β-actin (Millipore, Temecula, CA, USA). After washing, the membranes were further incubated for 2 h with 10,000-fold goat anti-mouse IgG (Calbiochem, Millipore, Billerica, MA, USA) or goat anti-rabbit IgG (Millipore) conjugated to horseradish peroxidase. The membranes were then washed and rinsed with ECL detection reagents (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK). The band images were photographed using ECL Hyperfilm (Amersham).