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Protein stability of CYP4B1.

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posted on 2015-09-10, 02:47 authored by Eva M. Schmidt, Constanze Wiek, Oliver T. Parkinson, Katharina Roellecke, Marcel Freund, Michael Gombert, Nadine Lottmann, Charles A. Steward, Christof M. Kramm, Vladimir Yarov-Yarovoy, Allan E. Rettie, Helmut Hanenberg

(A) Schematic outline of the lentiviral vector used for expression of CYP4B1. CMV: CMV promoter; SD: splice donor; LTR: long terminal repeat; SA: splice acceptor; RRE: Rev responsive element, cPPT: central polypurine binding tract; SFFV U3: U3 promoter of the spleen focus-forming virus; mcs: multicloning site; IRES: internal ribosomal entry site; neoR: neomycin phosphotransferase (nptII) resistance cDNA; EGFP: enhanced green fluorescent protein. (B) Protein half-life analysis of CYP4B1 isoforms in HepG2 cells. Protein half-life analysis was performed by adding 50 μg/ml cycloheximide (CHX) to the transduced and G418-selected HepG2 cells. The mean fluorescence intensity (MFI) of CYP4B1/EGFP fusion proteins was assessed at various time points by flow cytometry. For each construct, the mean ± SEM values are shown from at least three experiments.