Transcriptional up-regulation of the cyclin D2 gene and acquisition of new cyclin-dependent kinase partners in human T-cell leukemia virus type 1-infected cells
F Santiago, E Clark, S Chong, C Molina… - Journal of …, 1999 - Am Soc Microbiol
F Santiago, E Clark, S Chong, C Molina, F Mozafari, R Mahieux, M Fujii, N Azimi…
Journal of virology, 1999•Am Soc MicrobiolMATERIALS AND METHODS Tax and CREB expression vectors and protein purification.
Wild-type and mutant (M47) Tax proteins were overexpressed in Escherichia coli and
purified as described previously (30). Proteins were purified by nickel affinity
chromatography (Qiagen) followed by cation-exchange fast protein liquid chromatography
(HiTrap SP; Amersham Pharmacia Biotech)(23). For protein electroporation assays, E. coli-
expressed recombinant, purified Tax was electroporated as described previously (26) …
Wild-type and mutant (M47) Tax proteins were overexpressed in Escherichia coli and
purified as described previously (30). Proteins were purified by nickel affinity
chromatography (Qiagen) followed by cation-exchange fast protein liquid chromatography
(HiTrap SP; Amersham Pharmacia Biotech)(23). For protein electroporation assays, E. coli-
expressed recombinant, purified Tax was electroporated as described previously (26) …
MATERIALS AND METHODS
Tax and CREB expression vectors and protein purification. Wild-type and mutant (M47) Tax proteins were overexpressed in Escherichia coli and purified as described previously (30). Proteins were purified by nickel affinity chromatography (Qiagen) followed by cation-exchange fast protein liquid chromatography (HiTrap SP; Amersham Pharmacia Biotech)(23). For protein electroporation assays, E. coli-expressed recombinant, purified Tax was electroporated as described previously (26).
Protein transfection. Lymphocyte (CEM [12D7]) cells were grown to the mid-log phase of growth and processed for protein electroporation as described previously (26), with the modification that cells were electroporated at 230 V and plated in 10 ml of complete RPMI 1640 medium for 18 h prior to harvest.
American Society for Microbiology