Figure 3.

Proof-of-principle of Cross-and-Capture. (A) Detection of known protein complexes with Cross-and-Capture. Two diploids were examined, one expressing the bait and prey and the other expressing only the prey. Whole-cell extracts (WCEs) and pulldowns are probed with anti-V5. Pulldowns are additionally probed with anti-VSV. The origin-recognition complex (Orc1p–Orc5p), the chromatin assembly complex (Cac2p–Rlf2p), the casein kinase complex (Cka2p–Cka1p), DNA primase (Pri1p–Pri2p), the cyclin/TFIIH complex (Ccl1p–Tfb1p), and the Rad53p–Asf1p interaction are shown. For Rad53p–Asf1p interaction, growth for 1 h in 3 μg/mL 4NQO, is indicated by “+”. Note the band shift by phosphorylation (Rad53P–3×VSV) in presence of 4NQO. (B) Identification of the Shu1p–Psy3p complex. Pulldowns are done from diploids expressing Psy3 and Shu1 as both baits and preys. WCEs and pulldown are probed with anti-V5 and anti-VSV antibodies. Nonspecific background signals in the Western blots are indicated (*).