Fig. 6. The stable protein interactions between SAFB1/SAFB2 and Sam68/T-STAR are primarily mediated through their glutamate/arginine (ER)-rich regions. (A) Detection of protein interactions between SAFB1 and SAFB2 with T-STAR in vivo by immunoprecipitation. HEK293 cells were transfected with a construct encoding a T-STAR-FLAG fusion protein, and immunoprecipitated with affinity purified -SAFB1 and -SAFB2 antisera. Immunoprecipitated proteins were detected by probing with the FLAG antibody. (B) The ER-rich regions of SAFB1 and SAFB2 primarily mediate the protein interactions with T-STAR and Sam68, although a weaker interaction was identified with the glycine-rich region. SAFB1 and SAFB2 interact with the arginine and glycine-(RG) and tyrosine-rich (YD) containing C-terminal domains of Sam68 and T-STAR. Protein interactions were assayed by directed yeast two hybrid assays. The organisation of the interacting proteins are shown as cartoons, with the regions described in the text indicated, and the approximate extent of regions used in the assays illustrated underneath by double arrows. The strength of protein interaction in a filter lift assay is based on the rate at which colonies turned blue, and is indicated as strong (+++); medium (++); weak (+) and negative (–).