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Figure 4.

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Yjl122w/Alb1 is a physical partner of Arx1. (A) Alb1 is a two-hybrid prey of Arx1. YJL122W (ALB1) was found as prey ORF in a genomic two-hybrid screen performed with ARX1 as bait. The results are presented as in Fig. 1 A. (B) Rei1, Arx1, and Alb1 are associated with the same complexes. Rei1-, Arx1-, and Alb1-TAP–associated complexes were TAP purified. The Coomassie staining is displayed on the left. The presence of Arx1, Rei1, or Rlp24 in the TEV eluates was assessed by immunoblotting with specific antibodies against each of these proteins, as shown on the right, with the corresponding Ponceau red staining. (C) Alb1-TAP is associated with 60S particles. Whole cell extracts were prepared from a strain expressing Alb1-TAP, separated on a sucrose gradient, and analyzed as described in Fig. 1 C. Alb1-TAP was detected by Western blotting with PAP. (D) Arx1 and Alb1 interact directly in vitro. GST, GST-Rei1, GST-Alb1, and His6-Arx1 fusion proteins were produced in Escherichia coli BL21. Crude extracts containing the GST-tagged baits were mixed with equal amounts of crude extracts containing the His6-Arx1 putative prey and were purified on glutathione–Sepharose beads. The input samples as well as the eluted proteins were separated on a 10% polyacrylamide-SDS gel. The bait proteins were detected in the eluted fractions by Western blotting with antibodies against GST (top). His6-Arx1 was detected with antibodies against Arx1 in the fractions where it is coeluted (middle). As a charge control, His6-Arx1 was also detected in the input (bottom).

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