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. 2012 Aug 15;23(16):3240–3253. doi: 10.1091/mbc.E12-05-0339

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© 2012 Handa et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 7: — Pol ε is required for GINS to form complexes with Cut5, Cdc45, and Mcm6. Cdc20 protein was depleted as described in Figure 6. (A) Aliquots of HM3454 P_nmt81-cdc20-aid cdc45-myc 2xTIR1 cdc25-22 (tag-) and HM3450 P_nmt81-cdc20-aid psf2-flag cdc45-myc 2xTIR cdc25-22 without or with depletion (no depletion or Cdc20 depletion) were collected at 0 min and 100 min without addition of HU after G2/M release (see Figure 6) and treated with 1% formaldehyde. Proteins in WCE (lanes 1–6) and immunoprecipitates obtained with anti-FLAG (Psf2-FLAG IP, lanes 7–12) were analyzed by immunoblotting with anti-FLAG, anti-Sld5, anti-Psf1, anti-Psf3, anti-Cut5, anti-myc (Cdc45-myc), anti-Mcm6, or anti-tubulin antibodies. Triangles (◂) on the right of the panels indicate positions of the relevant proteins. Nonspecific protein bands are indicated by asterisks (*). It is noteworthy that thiamine-repressible proteins in WCE (lanes 1–4 in Sld5 panel) cross-reacted with the anti-Sld5 antibody (unpublished observation). The samples used for WCE (lanes 1–6) corresponded to 20% (Psf2-FLAG, Sld5, Psf1, Psf3, and tubulin), 1% (Cut5, Cdc45-myc), and 0.1% (Mcm6) of the proteins used for Psf2-FLAG IP. (B) Aliquots of HM4661 P_nmt81-cdc20-aid drc1-HA 2xTIR1 cdc25-22 (tag-) and HM4664 P_nmt81-cdc20-aid flag-dpb2 drc1-HA 2xTIR cdc25-22 without or with depletion (no depletion or Cdc20 depletion) at 0 min and 100 min after G2/M release were analyzed as in (A). Proteins in WCE (lanes 1–6) and immunoprecipitates obtained with anti-FLAG (FLAG-Dpb2 IP, lanes 7–12) were analyzed by immunoblotting with anti-FLAG, anti-Psf1, anti-Cut5, anti-Cdc45, or anti-Orc4 antibodies. Positions of the relevant proteins and a nonspecific protein are indicated by triangles (◂) and an asterisk (*), respectively. The samples used for WCE (lanes 1–6) corresponded to 20% (FLAG-Dpb2, Orc4) and 1% (Psf1, Cut5, and Cdc45) of the proteins used for FLAG-Dpb2 IP.