Goat polyclonal anti cox 2

The mice were sacrificed at the end of the experiment and the tumors were collected. The detection of the protein expression of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) was assessed using the western blot assay. The following primary antibodies were used: goat polyclonal anti-COX-2 and goat polyclonal anti-VEGFB antibodies (1:500 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The cancerous tissues were lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 100 mM Tris-HCl, 1% Tween-20, 1% sodium deoxycholate and 0.1% SDS) with 0.5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 10 μg/ml aprotinin and 1 μg/ml pepstatin. Proteins were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were then treated with the primary and secondary antibodies (goat anti-mouse IgG horseradish peroxidase-conjugated, 1:500 dilution, Santa Cruz Biotechnology, Inc.). Visualization was carried out using an enhanced chemiluminescence method (Amersham Bioscience, Boston, MA, USA). Subsequent to being stripped, the membranes were reprobed with β-actin (Oncogene, CN Biosciences, Inc., Darmastadt, Germany). The proteins were quantified using an Image Acquisition and Analysis System (Ultra-Violet Products, Upland, CA, USA).

Proteins were isolated from cell cultures by standard methods. A total amount of 30 μg of protein was loaded on a 6% or 12% SDS-PAGE gel. After electrophoresis, proteins were transferred to protran nitrocellulose membranes (Sigma-Aldrich, Madrid, Spain), and blots were probed with the indicated primary antibodies, as previously described [14 (link)]. The antibodies used were the following: rabbit polyclonal anti-C3 (Abcam, Cambridge, UK), mouse monoclonal anti-C/EBPβ (Abcam, Cambridge, UK), goat polyclonal anti-COX2 (Santa Cruz Biotechnologies, Mountain View, CA, USA), rabbit polyclonal anti-IL1β (Abcam, Cambridge, UK), rabbit polyclonal anti-α-tubulin (Sigma-Aldrich, Madrid, Spain) and secondary peroxidase conjugated antibodies (Jackson Immunoresearch, West Grove, PA, USA). Quantification was performed using Scion Image software (Scion Corporation, http://scion-image.software.informer.com/).