Figure 2

HDGF binds DNA. (A) HDGF binds DNA in vivo. ChIP was carried out as described in method. A specific HDGF polyclonal antibody was used to precipitate Hela chromatin, GFP antibody was used as negative control. Recovered DNA was amplified by PCR using specific primers. The PCR primers were derived from the human SMYD1 promoter as described in methods. The PCR products were analyzed by ethidium bromide staining of a 2% agarose gel. A primer set targeting a proximal site (-375 to -215) of the SMYD1 promoter was used as a negative control. The numbers on the right indict the location of amplicons in SMYD1 promoter. (B) HDGF binds DNA in vitro. 0.25 pmol 5' end biotin labeled probes for SMYD1 promoter (nucleotides -688 to -609) were incubated with 1 ug GST-HDGF fusion protein in the reaction buffer in the presence of either 100 × specific or non specific competitors (B) or various amount of sheared salmon sperm DNA (C) on ice for 30 min. The complex were resolved in 5% PAGE and transferred onto a nylon membrane. The biotin end-labeled DNA probe is detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescent substrate. (D) Different sources of HDGF bind to DNA. 1 ug of bacterial expressed GST-HDGF, His-HDGF-GFP and in vitro translated HDGF (without tag) were used for EMSA as described above.