Cyp2c9

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.

Liver and terminal ileum tissue samples were homogenized in RIPA (Beyotime Biotechnology, China), respectively. The total protein concentrations of the homogenates were measured with BCA reagent (BOSTER, China). An equal amount of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes by electroblotting. After blocking, the membranes were incubated with primary antibodies directed against human CYP1A2, CYP3A4, CYP2C9, P-glycoprotein, and GAPDH (Abcam, UK). Software ImageJ was used to analyze the Gray scale of the obtained images:
Gray value of each protein = gray value of target protein/gray value of internal reference protein.