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 file1 : Table S1. GAPDH served as an internal control and the results were analysed using the 2-ΔΔCt method.
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