Quantitative RT-PCR and Western Blot Analysis of mRNA and Protein Expression

For mRNA analysis, total RNA extraction for cDNA synthesis was conducted with the Taqman Reverse Transcription Reagent kit (TaKara-Bio, Kusatsu, Japan). Real-time quantitative PCR was conducted with different primer sequences (Table 1) using SYBR Green Master Mix (TaKaRa).Table 1

Primer sequences for real time RT-PCR

Target	Forward	Reverse
ACTB	TCTTCCAGCCTTCCTTCCT	AGCACTGTGTTGGCGTACAG
CAPRIN1	TCTCGGGGTGATCGACAAGAA	CCCTTTGTTCATTCGTTCCTGG
SLC2A1	TGTCTGGCATCAACGCTGTCTTC	TC CCTGCTCGCTCCACCACAAAC
HK2	CGACAGCATCATTGTTAAGGAG	CA GCAGGAAAGACACATCACATTT
HIF1A	AGTTCCGCAAGCCCTGAAAGC	GCAGTGGTAGTGGTGGCATTAGC
MYC	CGCCTCTTGACATTCTCCTC	GGACTATCCTGCTGCCAAGA
NUP160	GTTATCTGGCTGCTCTCAATTG	GTGCATTCTCCATCATGATTCC
NUP133	AGTACCTGTGGGCTGCTTCTCTAG	GCTCTGGTTGTCAGTCTGCTCAC
NUP155	CCGCTCCTCAGTCTCCCAGTG	GCTCATCCTTGGATCGCTGTGAC
METTL3	CCAGCACAGCTTCAGCAGTTCC	GCGTGGAGATGGCAAGACAGATG
WTAP	CTGACAAACGGACCAAGTAATG	AAAGTCATCTTCGGTTGTGTTG

Proteins were separated by SDS–PAGE followed by Western blot as described previously [30 (link), 31 (link)]. Anti- GAPDH (Cell Signaling Technology), anti-Caprin-1 (116 kDa, ProteinTech Group), anti-HIF1α (120 kDa, ProteinTech Group), anti-c-Myc (49 kDa, roteinTech Group), anti-WTAP (45 kDa, Santa Cruz Biotechnology), and anti-METTL3 (64 kDa, Abcam) were used as primary antibodies. HRP-conjugated anti-mouse and anti-rabbit IgG (Cell Signaling Technology) were used as secondary antibodies.