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Fig. 1. Protein-protein interactions within the SAS-I complex. (A) Sas4p interacted with Sas2p and Sas5p in the two-hybrid system as shown by growth or no growth of the reporter strain in the absence of histidine. GBD, Gal4 DNA-binding domain (pAS-BC); GAD, Gal4 activation domain (pACTIIst); see Table 2 for plasmid details. (B) Coimmunoprecipitation analysis of Sas5p-HA₃/myc₆-Sas2p, Sas4p-myc₉/Sas2p and Sas5p-HA₃/myc₆-Sas4p interactions. Bound proteins were analyzed on 12.5% SDS polyacrylamide gels, immunoblotted and probed with ${alpha}$ -myc or ${alpha}$ -Sas2 antibodies. 2 ${Delta}$ , SAS2 ${Delta}$ ; 4 ${Delta}$ , SAS4 ${Delta}$ ; 5 ${Delta}$ , SAS5 ${Delta}$ . (C) GST-pulldown assay to determine direct interactions within the SAS-I complex. Purified GST-Sas fusion proteins were immobilized on glutathione agarose beads and incubated with in vitro-translated radiolabeled SAS-I proteins (abbreviated 2, 4 and 5) or luciferase (L, negative control). An asterisk indicates degradation products of radiolabeled Sas4p. Washed beads were eluted with sample buffer, separated on 12.5% SDS polyacrylamide gels, and bands were visualized using a PhosphoImager.

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