Egfp n1 vector

HNF4α-overexpression plasmids were constructed by amplifying the HNF4α gene from the rat genome DNA by PCR with the forward primer 5′-ataagcttgacatggacatggctgacta-3′ (HindIII site underlined) and the reverse primer 5′-atggtaccctagatggcttcctgcttgg-3′ (KpnI site underlined). This 1401-bp rat HNF4α gene was inserted into the HindIII/KpnI sites of EGFP-N1 vector (BD Biosciences Clontech, Palo Alto, CA, USA) and confirmed by DNA sequencing, forming a recombinant HNF4α plasmid. The HNF4α plasmids were transfected to hepatic oval cells by Lipofectamin 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Two days post transfection, flow cytometry was used for sorting out EGFP-positive cells according to the method described previously [16 (link)], and the sorted cells were cultured in the presence of G418 (Invitrogen) antibiotic selection at 200 μg/ml for 18 days. HNF4α shRNA and negative control, noneffective shRNA were obtained from Santa Cruz (Dallas, TX, USA) and transfected to hepatic oval cells by Lipofectamin 3000 (Invitrogen) according to the manufacturer’s instructions. Two days post transfection, the cells were selected by Puromycin (Santa Cruz) antibiotic selection at 1 μg/ml for 18 days.

cDNA (pCMV6-Neo vector; OriGene, Rockville, MD) or CXCR7-green fluorescent protein (EGFP-N1 vector; BD Biosciences, San Jose, CA), a generous gift from Dr. Katherine Luker University of Michigan, Ann Arbor MI
[45 (link)]. EGFP-β-arrestin2 cDNA was a generous gift from Dr. V.R. Krishna Jala (Department of Immunology, University of Louisville, Louisville, KY). EGFR insert was cloned into a pLPCX plasmid under control of a CMV-IE promoter (Clontech, Mountainview, CA). Cells were transfected using Lipofectamine 2000 and analyzed for over expression by q-PCR and immunoblotting.