Fig. 4

HULC inhibitsthe expression and mature miR15a. a (left) RNA Immunoprecipitation (RIP) with anti-METTL3 followed by RT-PCR with pri-miR15a primers in Hep3B cell line. IgG RIP as negative control. RT-PCR for pri-miR15a as INPUT. (right) quantitive RIP analysis. b (left) RIP with anti-m6A followed by RT-PCR with pri-miR15a primers in liver cancer cells. IgG RIP as negative control. RT-PCR for pri-miR15a as INPUT. (right) quantitative RIP analysis. c (left) RIP with anti-DGCR8 followed by RT-PCR with pri-miR15a primers in Hep3B cell line. IgG RIP as negative control. RT-PCR for pri-miR15a as INPUT. (right) quantitative RIP analysis. d (left) RIP with anti-Droha followed by RT-PCR with pri-miR15a primers in liver cancer cells. IgG RIP as negative control. RT-PCR for pri-miR15a as INPUT. (right) quantitive RIP analysis. e Super-EMSA (gel-shift) with biotin-pre-miR15a probe and anti-Exportin5 antibody. The intensity of the band was examined by Western blotting with anti-Bioton. HistoneH3 as internal control. f Biotin-pre-miR15a pulldown followed by Western blotting with anti-Dicer, anti-ago2 Biotin as INPUT and β-actin as internal control. g Northern blotting analysis of miR15a in liver cancer cell Hep3B cell lines. h The real-time PCR detection of mature miR15a in liver cancer cells. U6 as internal control. i The real-time PCR detection of mature miR15a in Hep3B cell lines infected with rLV and rLV-miR15a respectively. Each value was presented as mean ± standard error of the mean (SEM).**, P < 0.01. j Westernbloting with anti-P62, anti-Jun, anti-Notch, anti-PTEN, anti-mTOR. β-actin as internal control