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. 2017 Apr 13;8(30):49093–49109. doi: 10.18632/oncotarget.17095

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Copyright: © 2017 An et al.

This article is distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use and redistribution provided that the original author and source are credited.

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(A) Super-EMSA(gel-shift) with biotin-Pim1 promoter probe and anti- P21(WAF1/Cip1) antibody. The intensity of the band was examined by Western blotting with anti-Biotin.HistoneH3 as internal control. (B) Chromatin Immunoprecipitation(CHIP) with anti- P21(WAF1/Cip1) followed by PCR with Pim1 promoter primers in liver cancer cells Hep3B cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-JMJD2A, rLV-JMJD2AΔ, pCMV6-AC-GFP-JMJD2A plus miR372 inhibitor, rLV-JMJD2AΔ plus miR372 inhibitor, respectively. IgG CHIP as negative control. Pim1 promoter as INPUT. (C) Chromatin Immunoprecipitation(CHIP) with anti-RNApolII followed by PCR with Pim1 promoter primers in liver cancer cells Hep3B cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-JMJD2A, rLV-JMJD2AΔ, pCMV6-AC-GFP-JMJD2A plus miR372 inhibitor, rLV-JMJD2AΔ plus miR372 inhibitor, respectively. IgG CHIP as negative control. Pim1 WAF1/Cip1 promoter as INPUT. Antisense-mature miR372 as miR372 inhibitor. (D) The assay of Pim1 promoter luciferase activety. Each value was presented as mean ± standard error of the mean (SEM).**P < 0.01. (E) Western blotting analysis with anti- anti-Pim1, anti-CDK2, anti-MEKK1, anti-MEKK4 in liver cancer cells Hep3B cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-JMJD2A, rLV-miR372, rLV-JMJD2AΔ, pCMV6-AC-GFP-JMJD2A plus miR372 inhibitor, rLV-JMJD2AΔ plus miR372 inhibitor, respectively. β-actin as internal control. (F) Western blotting analysis with anti-Pim1,anti-P21(WAF1/Cip1) in liver cancer cells Hep3B cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-JMJD2A, pCMV6-AC-GFP-JMJD2A plus pcDNA3.1-P21(WAF1/Cip1), respectively. β-actin as internal control. (G) The assay of Pim1 methylation through Co-Immunoprecipitation(IP) with anti-me followed by Western blotting with anti-Pim1 in liver cancer cells Hep3B cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-JMJD2A, pCMV6-AC-GFP-JMJD2A plus pcDNA3.1-P21(WAF1/Cip1), respectively. IgG IP as negative control. INPUT refers to Western blotting with anti-pim1.