Plasmid construction and mutagenesis

Expression plasmids for epitope-tagged E2F1, SHP and EID1 have been previously described (15 (link), 19 (link)). The human HMGB2 3′UTR containing the predicted miR-127 binding site was cloned into pMIR-Reporter (Invitrogen) using primers 5′-TCAGACTAGTCAAGTGCAGCTCAATACT-3′ and 5′-ACCAAGCTTCACCTGAGGAACAATTTA-3′. Mutations in pMIR-Reporter-HMGB2-3′UTR were generated with a SiteMutant Kit using primers 5′-TGAATTCAAGTGCAGCTCAATACTAGCTAGTGTATAAAAACTGTACAGATTTTTGTATAG-3′ and 5′-CTATACAAAAATCTGTACAGTTTTTATACACTAGCTAGTATTGAGCTGCACTTGAATTCA-3′. The plasmids were confirmed by sequencing. The expression plasmid pTarget-miR-127 and pTarget-miR-433 were generated as described (11 (link)). Plasmid pCAG-Myc-Oct3/4-IP, pCAG-HA-Sox2-IP were gifts from Dr. Shinya Yamanaka. pCDNA3.1 Flag mHMGB2 and reporter gene plasmid 6xO/S luc were gifts from Drs. Yasuhiko Kawakam and Lisa Dailey, respectively.