HA- and FLAG-tagged KLF15 were constructed by PCR using untagged human KLF15 purchased from the Arizona State University plasmid repository as a template using and following primers: HA-KLF15-Forward-5′-GGG CCC GGA TCC ATG TAC CCC TAC GAC GTG CCC GAC TAC GCC GTG GAC CAC TTA CTT -3′; HA-KLF15-Reverse-5′GGG CCC CTC GAG CTA GTT CAC GGA GCG CAC GG-3′- -3′. 135 amino acids at KLF15 C-terminus corresponding to zinc-finger domain in KLF15 were truncated using following primers: Forward:5′-GGG CCC GAA TTC CTA AAT GCG CAC AAA CTT GGA GGG C-3′; Reverse:5′-GGG CCC GAA TTC CTA GTT CAC GGA GCG CAC GG-3′.
The PCR product was cloned into pcDNA3.1 using directional topo cloning (Invitrogen) Adenoviruses encoding HA and FLAG tagged KLF15 was constructed by subcloning into entry PENTR1a vector using and restriction enzymes followed by LR recombination with destination vector PDEST. The HDAC2-PDEST DNA was digested with PacI, ethanol precipitated and transfected into 293 HEK cells. After cytopathic effect (CPE), adenoviruses were collected and purified via three freeze-thaw cycles and a Millipore adenovirus purification Kit.
The PCR product was cloned into pcDNA3.1 using directional topo cloning (Invitrogen) Adenoviruses encoding HA and FLAG tagged KLF15 was constructed by subcloning into entry PENTR1a vector using and restriction enzymes followed by LR recombination with destination vector PDEST. The HDAC2-PDEST DNA was digested with PacI, ethanol precipitated and transfected into 293 HEK cells. After cytopathic effect (CPE), adenoviruses were collected and purified via three freeze-thaw cycles and a Millipore adenovirus purification Kit.