Fig. 3.

Self-interactions of Cdc45 and GINS. a, b Cdc45-5FLAG was precipitated via M2 beads from WCE (a), non-CHR or CHR (b) fractions of the CDC45-5FLAG/pCDC45-13MYC cells (Strain LL85-1, Table S1). Co-precipitated proteins were detected via immunoblots against the indicated antibodies. Mcm10 was also probed to validate the fractionation of non-CHR and CHR-associated proteins given the fact that it binds to Cdc45 exclusively in the context of chromatin. In, input. c Cdc45-3HA-EPEA was precipitated via a C-tag affinity matrix from WCE of the CDC45-3HA-EPEA/pCDC45-13MYC cells (Strain LL111-1, Table S1). Co-precipitated proteins were detected as above. d, e Pfs2-5FLAG was precipitated via M2 beads from WCE (d), non-CHR or CHR (e) fractions of the PSF2-5FLAG/pPSF2-7MYC cells (LL67-1, Table S1). The precipitates were subjected to immunoblotting. Cross-reacting bands (d) are labeled by asterisks. Mcm2 was also probed to validate the fractionation of non-CHR and CHR-associated proteins. f GST pull-down analysis of Cdc45 self-interaction. Recombinant 6His-Cdc45 was incubated with GST-Cdc45 or GST alone in the presence of glutathione beads. After separation by SDS-PAGE, the bound proteins were analyzed via Coomassie Brilliant Blue staining and immunoblotting, respectively. g Yeast two-hybrid analysis of the self-interaction of the GINS subunits. The yeast cells expressing the indicated activation domain (AD)- and DNA binding domain (BD)- fusion proteins were grown at 30 °C on either SC-Trp-Leu or SC-Trp-Leu-His plates