Cox 1

Proteins within tumour tissues and HUVEC cells were extracted using a Boster Kit (Bosterbio, CA, USA) according to the manufacturer’s instructions. Western blotting was performed as previously described [20 (link)]. The antibody, which was raised against the Cyp2c44’s IGRHQPPSMKDKMKC peptide (GenScript), was generated according to previous studies [13 (link), 16 (link)]. Other antibodies used in this study are as follows: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bosterbio), β-actin (Bosterbio), Cyp2c9 (Abcam, Cambridge, UK), COX1 (Santa Cruz, CA, USA), COX2 (Santa Cruz), Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) (Santa Cruz), ERK1/2 (Santa Cruz), P-AKT (Abcam), AKT (Abcam), and CD31 (Abcam).
For qRT-PCR, RNA from tumours was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and reverse-transcribed using the M-MLV First-Strand cDNA Synthesis Kit (Invitrogen) [15 (link)]. The mRNA levels of target genes were quantified by qRT-PCR using Power SYBR Green PCR Master Mix (Invitrogen) with the primers listed in Additional file 1: Table S1. GAPDH served as an internal control and the results were analysed using the 2^-ΔΔCt method.

Ascending aortic medial tissue from 6-mo-old wild-type (male n = 6; female n = 6) and Marfan (male n = 6; female n = 6) mice was homogenized using a bullet blender, ø 0.9–2 and 3 mm stainless steel beads (Next Advance, NY, USA) in 300 μl radioimmunoprecipitation assay buffer (RIPA, 10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 7.5, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium desoxycholate, 0.1% SDS, 140 mM NaCl) and protease inhibitors. Protein concentrations were assessed using a colorimetric assay for protein concentration (Biorad, CA, USA). Twenty micrograms of protein per sample were analyzed by 10% (v/v) SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies to COX-1 (Santa Cruz, CA, USA), COX-2, eNOS, (p)eNOS (BD Transduction Laboratories, CA, USA), iNOS (Thermo Fisher, MA, USA) and actin (Sigma Aldrich, MO, USA) were incubated overnight in TBS 1% BSA. Protein bands were revealed using secondary IgG HRP conjugates (Promega, WI, USA) and Luminol Reagent together with Hyperfilm (Amersham Pharmacia Biotech, Uppsala, Sweden). Band intensities were measured by densitometry scanning using ImageJ software (National Institute of Health, Bethesda, MD, USA). Band intensities were relativized against actin as a loading control and the values of all groups were normalized for WT male average values.

We used the following antibodies: anti-Ionized calcium binding adapter molecule 1 (Iba-1, Western blot (WB); 1: 1000, Immunohistochemistry (IHC); 1:500, 019-19741, Wako, Osaka, Japan), anti-cyclooxygenase-1 (COX-1, WB; 1: 200, sc-19998, Santa Cruz Biotechnology, CA), anti-COX-2 (WB; 1: 100, sc-376861, Santa Cruz Biotechnology, CA), anti-L-PGDS (WB; 1: 500, PA1-46023, Thermo Fisher Scientific, Tokyo, Japan), anti-H-PGDS (WB; 1: 1000, PA5-24347, Thermo Fisher Scientific, Tokyo, Japan), anti-doublecortin (DCX, WB; 1: 1000, IHC; 1: 2000, ab18723, Abcam, Cambridge, MA), anti-Ki67 (IHC; 1: 100, NB500-170, Novus Biologicals, Inc., Littleton, CO), anti-superoxide dismutase 2 (SOD2, WB; 1: 1000, 13194, Cell Signaling Technology, Beverly, MA), anti-14-3-3ς (WB; 1: 1000, 7413, Cell Signaling Technology, Beverly, MA), anti-synaptophysin (WB; 1: 20000, ab32127, Abcam, Cambridge, MA), anti-postsynaptic density protein 95 (PSD95, WB; 1: 250, 610495, BD Biosciences, San Diego, CA), anti-Nuclear factor erythroid 2-related factor 2 (Nrf2, WB; 1: 500, Proteintech, Chicago, MA), anti-α-tubulin (WB; 1: 4000, T5168, Sigma-Aldrich, Deisen-hofen, Germany), and anti-GAPDH (WB; 1: 1000, ABS16, Millipore, Billerica, MA).