Fig 1: miR-1307 inhibits the expression of KDM3A and KDM3B by reducing METTL8 in liver cancer cells(A) RNA immunoprecipitation (RIP) with anti-METTL8 was performed. RT-PCR was used to detect KDM3A and KDM3B mRNA. IgG RNA immunoprecipitation was used as negative control.(B) RT-PCR was used to detect KDM3A and KDM3B. ß-actin was used as internal reference gene.(C) (a) The total protein was extracted, and the expression of KDM3A and KDM3B was detected by western blotting. ß-actin was used as the internal reference gene. (b) Gray scan analysis of positive bands. Each experiment was repeated three times. The values of each group were expressed as mean ± SD (n = 3); * *p < 0.01, *p < 0.05.(D) The expression of METTL8 was analyzed by western blot. ß-actin was used as the internal reference gene.(E) RT-PCR was used to detect KDM3A and KDM3B. ß-actin was used as internal reference gene.(F) (a) The expression of KDM3A and KDM3B was detected by western blotting. ß-actin was used as the internal reference gene. (b) Gray scan analysis of positive bands.
Fig 2: miR-1307 targets METTL8 and inhibits METTL8 expression(A) Total RNA was extracted, and northern blotting was used to detect miR-1307. U6 as internal reference genes.(B) qRT-PCR was used to detect the mature miR-1307. Each experiment was repeated three times. The values of each group were expressed as mean ± SD (n = 3); * *p < 0.01, *p < 0.05.(C) Bioinformatics was used to analyze the seed sequence of mature miR-1307 binding to 3'UTR of METTL8 mRNA.(D) The activity of pMirtarget-METTL8 3'-UTR-Luc reporter gene was detected. Each experiment was repeated three times. The values of each group were expressed as mean ± SD (n = 3); * *p < 0.01, *p < 0.05.(E) The activity of pMirtarget-METTL8 3' UTR(mutant) Luc reporter gene.(F) RT-PCR was used to detect METTL8. ß-actin was used as the internal reference gene.(G) Quantitative RT-PCR analysis. Each experiment was repeated three times. The values of each group were expressed as mean ± SD (n = 3); * *p < 0.01, *p < 0.05.(H) Western blotting was used to detect METTL8. ß-actin was used as internal reference gene.(I) Gray scan analysis of positive bands. Each experiment was repeated three times. The values of each group were expressed as mean ± SD (n = 3); * *p < 0.01, *p < 0.05.
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