Semi-quantitative reverse transcriptase PCR (RT-PCR) and quantitative real-time PCR (qPCR)

RT-PCR and qPCR are described in our previous work ¹⁷. Total RNA was extracted from cells using Trizol reagent (Invitrogen, USA) as described by the manufacturer. The RNA was then converted to cDNA via reverse transcriptase using a cDNA synthesis kit (TaKaRa, Japan) according to the manufacturer's instructions. For RT-PCR, 1 μL of the reverse transcription product was amplified by Ex Taq (TaKaRa, Japan) using primers pcDNA3.1-T7-FP, Axl-E9-FP, and Axl-E11-RP (Table S1). The inclusion and exclusion forms of exon10 of Axl were detected by RT-PCR. The positions of these two forms are depicted on 2% agarose gels. Image J software was then used for grayscale analysis by the ratio of exon skipping means the grayscale value of Axl-L/ Axl-S. For qPCR, synthesized cDNA was used in qPCR experiments with the SYBR Green mix (TaKaRa, Japan) and analyzed with a Bio-Rad Real-Time detection system (Bio-Rad, USA). The relative expression of target genes was calculated and normalized using the 2^-ΔΔCt method. The primers are shown in table S1.