Anti cox 2

Western blots were performed as described in our previous studies [14 (link),36 (link)]. Specific primary antibody anti-COX-2 (1:600, Santa Cruz Biotechnology) or anti-PGE2 (1:700; Bioss Antibodies) was mixed in 1× PBS, 5% w/v nonfat dried milk, and 0.1% Tween-20, and incubated at 4 °C, overnight. After that, blots were incubated with the peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research) for 1 h at room temperature. To verify the equal amounts of protein, membranes were also incubated with the antibody against beta actin (1:1000; Santa Cruz Biotechnology). Signals were detected with enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate, Pierce). The relative expression of protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software and standardized to β-actin levels. Images of blot signals were imported to analysis software (Image Quant TL, v2003).

Immunohistochemistry was performed as reported previously (Hoare et al., 2016 (link)). Formalin fixed paraffin-embedded mouse tissues were stained with the following antibodies: anti-Cox2 (as above); anti-NRAS (Santa Cruz, sc-31,1:100); anti-p21 (BD, 556431, 1:50); anti-ki67 (Bethyl, IHC-00375, 1:1000); anti-Ly6C (Abcam, ab15627, 1:400); anti-Cd11c (Cell Signaling, 97585, 1:350); anti-Cxcl1 (Abcam, ab86436, 1:100); anti-PGE₂ (Abcam, ab2318, 1:100); anti-Foxp3 (eBioscience, 14-5773, 1:100); after proteinase K digestion (Ly6C) or heat-induced epitope retrieval in citrate (pH6) or Tris-EDTA (pH9) buffers before visualization using the DAKO Envision kit according to manufacturer’s instructions and counterstaining with hematoxylin. For fluorescent labeling we utilized appropriate fluorochrome-tagged secondary antibodies (Life Technologies). For EdU staining (ThermoFisher C10638), the same protocol was followed, with the following extra step: after antigen retrieval, 3% BSA washes in PBS were performed twice, and CliCK-iT reaction cocktail was added for 30 min following the manufacturer’s instructions.
All slides were scanned on a Leica AT2 at 20x magnification and a resolution of 0.5 μm/pixel. Following digitization, image analysis was performed as described previously using HALO (Indicalabs) (Hoare et al., 2016 (link)).

Proteins from BV2 cells were isolated according to standard techniques, separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene fluoride membrane (PVDF, Millipore, Bedford, MA). Blots were then probed for anti-COX2 (Santa Cruz Biotechnology, Dallas, TX), anti-iNOS (BD Biosciences, San Jose, CA), anti-p38, anti-p-p38, anti-JNK, anti-p-JNK, anti-ERK, or anti-p-ERK (Cell Signaling, Danvers, MA), after which they were incubated with secondary antibody conjugated with horseradish peroxidase. The intensity of chemiluminescence was measured using an ImageQuant LAS 4000 apparatus (GE Healthcare Life Sciences, Buckinghamshire, UK). The membrane was then reprobed with an anti-β-actin antibody (Sigma-Aldrich) as an internal control.

Ginkgolic acid C 17:1 (GAC 17:1) (95% purity) and ginkgolic acid C 15:1 (GAC 15:1) (98% purity), anti-STAT3, anti-SHP-1, anti-PTEN, anti-Bcl-2, anti-Bcl-xL, anti-survivin, anti-IAP-1, anti-COX-2, anti-MMP-9, anti-MMP-2, anti-caspase-3, anti-PARP, anti-β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris base, glycine, NaCl, sodium dodecylsulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640, MEM, and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Annexin V was obtained from BD Biosciences (Palo Alto, CA, USA). Anti-p-STAT3 (Tyr705), anti-p-JAK1 (Tyr1022/1023), anti-JAK1, anti-p-src (Tyr416), anti-src, anti-cyclin D1, and anti-cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). pMXs-gw (#18656) and pMXs-STAT3C (#13373) were obtained from Addgene (Cambridge, MA, USA).

The 4-hydroxynonenal was obtained from Biomol (Plymouth Meeting, PA, USA). Dulbecco's modified essential medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO Invitrogen (Carlsbad, CA, USA). The following antibodies, anti-COX-2, anti-Lamin A/C, and anti-beta actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Pim-2 and myc-tagged antibodies were from Millipore (Billerica, MA, USA). The pp70S6K, p70S6K, p4EBP1, 4EBP1, pBad, Bad, NF-κB (p65), and GAPDH antibodies were from Cell Signaling Technology (Danvers, MA, USA). Other chemicals were of the highest purity available.