Pcmv6 hsix1 myc ddk

A 222 bp fragment corresponding to the region -13,982 to -13,761 from the Cidea transcription start site (TSS) was PCR-amplified from mouse genomic DNA and cloned into pGL3-Basic plasmid (Promega) to yield pGL3-Cidea-Enh1. For the luciferase assay, HEK293 cells were transfected with pGL3-Cidea-Enh1 (or equal amounts of pGL3-Basic) and pCMV6-hSix1:myc:DDK from OriGene (RC203465) (or equal amounts of pCMV6:myc:DDK) using Lipofectamine 2000 (Invitrogen). 48h after transfection cells were lysed and Firefly luciferase activity was measured on a GloMax Multi luciferase reader using the Dual-Glo luciferase assay kit (Promega) following the manufacturer’s recommendations.

HEK293 cells were transfected using Lipofectamine 2000 (Invitrogen) with the following plasmids: pCMV6-hSix1:myc:DDK from Origene (RC203465); pcDNA3.1-Cebpa:HA, a kind gift from Dr. D. Tenen; pcDNA3.1-mCebpb from Addgene (12557); pLenti-EF1ap-mEbf2:tGFP (Origene). Three days after transfection, cells were harvested and lysates were prepared as for Western blots except that the lysis buffer contained 50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP40, and proteinase inhibitor mix (Roche, 11 873 580 001). 500 μg of total proteins were pre-cleared with 10 μl protein G beads (Sigma, P3296) and incubated overnight with 10 μl anti-FLAG M2 affinity gel at 4°C on a rotator (Sigma, A2220). The gel was washed twice each with lysis buffer, RIPA buffer containing 0.1% SDS, RIPA buffer containing 0.1% SDS and 1% NP-40. Precipitated proteins were eluted using SDS loading buffer (95°C, 5 min) and separated on PAGE gels for Western analysis.