FIG 3.

hnRNP K binds to the single-stranded poly(C) motif directly. (A) Schematic description of the motif analysis. Proximal promoter regions of 1,056 genes were analyzed using the database from reference 29. (B) Motif analysis showed that the CCC motif was frequently found along with a CCT motif. (C) Coomassie blue staining showed that one thick band was bound onto the forward poly(C) motif oligonucleotide, which was identified as hnRNP K by mass spectrometry. In contrast, there were no prominent proteins detected at the complementary poly(C) motif oligonucleotide. (D) Purification of GST and GST-hnRNP K was also carried out for pulldown assay. GST-hnRNP K was detected at the wild-type oligonucleotide but not at the mutant oligonucleotide, which demonstrates that hnRNP K is directly bound to the poly(C) motif. (E) To identify the effect of knockdown of hnRNP K on WT(Short) and the poly(C) mutant, a promoter assay was conducted with siRNA transfection. It showed a significant decrease only for WT(Short) promoter activity (n = 3; n.s., not significant; *, P < 0.05 by two-way ANOVA followed by Sidak’s multiple-comparison test). Error bars indicate SEM.