Fig. 1. Interaction between OBF-1 and Siah-1 in yeast and in mammalian cells. (A) β-galactosidase activity in the yeast strain EGY48 containing a lacZ reporter under the transcriptional control of multimerized lexA binding sites and different combinations of expression plasmids as indicated. Importantly, VP16-Siah-1 only activates transcription in cells also expressing Lex-OBF1–114. The relative β-galactosidase activity of the different strains was normalized for protein level. (B) Co-immunoprecipitation assay. 293T cells were transfected with an expression vector for myc-Siah-1 (500 ng or 1 µg), together with expression vectors for POU-1-HA or OBF-1-HA, as indicated. Forty-eight hours after transfection cells were treated for 6 h with 500 nM MG262 in order to stabilize Siah-1. After cell extract preparation, HA-tagged proteins were immunoprecipitated with the 12CA5 mAb. The precipitate was analyzed by SDS–PAGE and blotted with the 9E10 mAb to detect potential association of Siah-1. Expression of POU-1 or OBF-1 was detected by reprobing the membrane with the 12CA5 mAb (lower part).