Verification of yeast two-hybrid interaction of parafibromin with actinin. (A) GST or GST-fusion proteins used for in vitro binding assay were analyzed by SDS-PAGE and stained with Coomassie Blue. On the left are shown molecular weights of the protein standards in kilodaltons. (B) GST pull-down assay: GST or GST-fusion proteins coupled to glutathione sepharose beads were incubated with whole cell protein extracts from HEK293 cells transfected with plasmid expressing parafibromin-myc-his. The beads were washed thoroughly and the bound parafibromin was detected by western blotting (WB) with an anti-myc antibody. Input corresponds to 1/40th of the amount of protein extracts used for the pull-down assay. (C) Co-immunoprecipitation assay: Whole cell protein extracts from HEK293 cells transfected with plasmids expressing parafibromin-myc-his or menin-myc-his alone or together with FLAG-actinin-2 or FLAG-actinin-3 were immunoprecipitated with a rabbit anti-myc antibody. The immunoprecipitates were analyzed by western blotting (WB) with a mouse anti-myc (to detect parafibromin) or anti-FLAG (to detect actinin) antibody. Input panels show portions of protein extracts corresponding to 1/40th of the amount used for each immunoprecipitation (IP), probed with anti-myc or anti-FLAG antibody.