Rabbit anti cox 2 polyclonal antibody

Proteins from total cell lysates were separated by 10% SDS‐PAGE and transferred electrophoretically onto Immobilon‐P polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The membranes were probed with rabbit anti‐COX‐1 polyclonal antibody (1:1,000; Cayman Chemical), rabbit anti‐COX‐2 polyclonal antibody (1:200; Cayman Chemical), rabbit anti‐p38 MAPK monoclonal antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA, catalogue no. 9212S), rabbit anti‐p‐p38 MAPK (T180/Y182) monoclonal antibody (1:1,000; Cell Signaling Technology, catalogue no. 9211S), mouse anti‐Bcl‐2 monoclonal antibody (1:500; Proteintech, Rosemont, IL, USA), mouse anti‐RhoA monoclonal antibody (1:500; Santa Cruz Biotechnology, Ann Harbor, MI, USA) or mouse anti‐tubulin monoclonal antibody (1:5,000, Cell Signaling Technology) followed by either anti‐rabbit or anti‐mouse IgG secondary antibodies conjugated to horseradish peroxidase (1:4000; Proteintech) and detection with the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA). The films were scanned by the ImageQuant LAS 4000 mini biomolecular imager (GE Healthcare, Chicago, Illinois, USA). The relative protein density was quantified by ImageJ 1.44 (NIH, Bethesda, Maryland, USA).

RAW 264.7 cells were grown in Poly-D-lysine-coated glass dishes for 24 h then treated with LPS and compounds and extracts tested as described above. After treatment, cells were fixed with cold 4% (w/v) paraformaldehyde for 20 min, washed in PBS, and then incubated for 15 min with 0.1% (w/v) TritonX-100 and 3% Bovine Serum Albumin (BSA). Thereafter, the cells were incubated with Rabbit anti-COX-2 polyclonal antibody (1:200) (Cayman, Ann Arbor, MI, USA, catalog number 160126) at 4 °C overnight, followed by the fluorescent secondary antibody: AlexaFluor 586 goat anti-rabbit (1:333) (Invitrogen, Carlsbad, CA, USA). Nuclei were also counterstained with DAPI dye (Sigma-Aldrich, Milan, Italy). Microscopic analysis was performed with an Olympus BX63 microscope equipped with a Metal Halide Lamp (Prior Scientific Instruments Ltd., Cambridge, UK) and a digital camera, Olympus XM 10 (Olympus, Milan, Italy).

Cells were lysed in a 300 µL radioimmunoprecipitation assay buffer (RIPA) (Sigma-Aldrich, Milan, Italy). Total protein content was measured by using the Bio-Rad DC protein assay kit (Bio-Rad, Milan, Italy). Equal aliquots (30 μg) of proteins were applied to a nitrocellulose membrane (Millipore, Burlington, VT, USA) and allowed to dry for 30 min at RT. After blocking with 6% nonfat dry milk for 1 h at RT, the membranes were incubated overnight at RT with the Rabbit anti-COX-2 polyclonal antibody (1:200) (Cayman Chemical, MI, USA, catalog number 160126) followed by incubation with anti-rabbit IgG horseradish peroxidase-linked antibody (Cell Signaling, Danvers, MA, USA), 1:4000 for 1 h at RT. Chemiluminescence was developed by using the Immobilon Horseradish Peroxidase Substrate (Merck Millipore, Darmstadt, Germany), and immunoreactive spots were quantified using Quantity-One software (Bio-Rad Laboratories S.r.l., Milan, Italy).