Anti v5 agarose

Wild-type Mcm2–7/Cdt1 and ORC complexes were purified as described previously (Kang et al., 2014 (link)). Wild-type Cdc6 was purified as described in Frigola et al. (2013) (link). DDK, S-CDK, Sld3/7, Cdc45, Sld2, Dpb11, GINS, Mcm10, Polymerase epsilon, Polymerase alpha/primase, Polymerase delta, RPA, Ctf4, RFC, PCNA, Mrc1, Csm3-Tof1, and Topo II were purified as described in Lõoke et al. (2017) (link). Mutant Mcm2-7/Cdt1 complexes were purified as described in Kang et al. (2014) (link) with the following modifications. For each Mcm2-7 mutant complex, the corresponding wild-type proteins were epitope-tagged with either c-Myc or V5. In the strains expressing the Mcm2 Δ2–177 and Mcm6 Δ2–105, the wild-type MCM2 and MCM6 genes were tagged with c-Myc, to allow the endogenous 13Myc-tagged Mcm2 or Mcm6 subunits to be depleted by incubating with anti-c-Myc agarose (Sigma) before applying the Mcm2-7 mutant complex to a Superdex 200 gel filtration column. In strains expressing mutant Mcm4 protein, the wild-type MCM4 gene was tagged with V5. Mcm2-7 complexes containing the endogenous V5-tagged Mcm4 subunits were depleted by incubating with anti-V5 agarose (Sigma) before application to a Superdex 200 gel filtration column. Yeast strains and plasmids used are listed in Supplementary file 1 - tables 2 and 3, respectively.

CHO-DSP1 cells were transfected as described in the previous section with pCAGGS-gB vectors carrying the N-terminal mutations, pME18s-gH[V5] and pCDNA3.1-gL (1.6μg of each vector). To determine the specificity of the V5 immunoprecipitation of the gB/gH-gL complex, CHO-DSP1 cells were transfected pCAGGS-gB, pCDNA3.1-gL, pME18s-gH[WT] or pME18s-gH[V5], and the previously well characterized pBud-gE/gI vector [36 (link)] (1.5μg of each vector). At 24 hours post transfection cells were lysed with glycoprotein lysis buffer and snap frozen in liquid nitrogen and stored at -20°C. The gB/gH-gL complexes were immunoprecipitated with anti-V5 agarose (Sigma). Wash steps, protein elution and SDS-PAGE were performed as outlined in the previous section. Western blots were performed using either mouse anti-V5 tag (Bio-Rad), 746–868 rabbit poly clonal IgG, mAb 93k or mouse anti-gE (Millipore). The anti-gE mAb is very sensitive and can detect low concentrations of gE [36 (link),80 (link)–82 (link)], critical for demonstrating the specificity of the gB/gH-gL immunoprecipitation.

Expression plasmids were transfected into HEK293, OVCAR3, or MCF7 cells using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 hr post-transfection for co-immunoprecipitation experiments. Cells were lysed in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP40 supplemented with protease inhibitor cocktail (Thermo Scientific)). Lysates were then incubated with either anti-V5 agarose (Sigma, St. Louis, MO), or anti-FLAG M2 affinity gel (Sigma, St. Louis, MO). After five washes (3 times with lysis buffer and 2 times with TBS) precipitates were resuspended in Laemmli sample buffer containing 5% β-mercaptoethanol.