His tag

Pull-down experiments with His-tagged proteins mixed with GST, GST-Survivin or GST-Survivin mutants were carried out as described (78 (link)). For streptavidin-binding experiments, 50 μg of biotinylated peptides were bound to 25–50 μl of a 50:50 slurry of streptavidin agarose (Sigma-Aldrich), mixed with recombinant proteins or cell lysates, and analyzed by Western blotting. Immunoprecipitation using primary antibodies (10 μg/mL), and 200–400 μg of total or fractionated cell extracts were carried out as described (80 (link)). The following antibodies to Cdk1, PT14/PY15-Cdk1, 14–3-3 β, Lamin B (all from Santa Cruz Biotechnology), Cyclin B1 (PharMingen), Survivin (NOVUS Biologicals), α-tubulin (clone B-5–1-2) (Sigma-Aldrich) and His-tag (Invitrogen) were used. Protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences).

The samples derived from PA14 WT and its derived mutation strains were separated and transferred to nitrocellulose membranes (GE-Healthcare, Pittsburgh, PA). Membranes were incubated with mouse monoclonal antibody against GFP (Biolegend, San Diego, CA) and His-tag (ThermoFisher Scientific, Waltham, MA) at 1:5000 for overnight at 4 °C with primary antibodies^{44 (link),45 (link)}. After washing, adding corresponding secondary antibodies for 1.5 h. After washing five times with washing buffer, the protein bands were visualized by chemiluminescence.

Antibodies against Mcm10, Mcm2 and Mcm2-phosphoserine-164-phosphoserine-170-phosphoserine, Cdc45, GINS and Sld3 were supplied by Open Biosystems (we supplied the antigens). Crude serum was purified against immobilized antigen to remove nonspecific antibodies. The specificity of each antibody was analyzed by western blot of purified proteins and yeast extract. Antibodies directed against RPA (Thermo Scientific), FLAG epitope (Sigma), Arp3 (Santa Cruz Biotechnology) and His Tag (Thermo Scientific) were commercially purchased.

Treated cells were harvested in NP40 lysis buffer (1% NP40 + 50 mM Tris-HCl + 150 mM NaCl) and fractionated^{21 (link)}. Briefly, samples underwent two rounds of sonication (60 s, 1 s pulse on/off at 10% power) and centrifugation, prior to re-suspending the insoluble pellet in fresh NP40 lysis buffer. Western blotting used antibodies to detect GFP (Santa Cruz Biotechnology, sc-9996, 1:1000), GAPDH (Santa Cruz Biotechnology, sc-47724, 1:4000), Histone H3 (Cell Signaling Technology, 9715, 1:10,000), hnRNPA1 (ThermoFisher Scientific, PA5-19431, 1:1,000), hnRNPA0 (ThermoFisher Scientific, PA5-57722, 1:1000), and HIS-tag (ThermoFisher Scientific, MA1-21315, 1:5000). α-mouse HRP (ThermoFisher Scientific, A16011, 1:10,000) or α-rabbit HRP (ThermoFisher Scientific, A16023, 1:10,000) were applied prior to detection on the Amerhsam Imager 600 (GE Healthcare Life Sciences). Relative intensities were calculated as the average pixel intensity of heat shocked insoluble fraction bands compared to the average pixel intensity of the cell lysate bands, normalized against background. Intensity measurements were made using ImageJ 1.52a (National Institutes of Health). For each sample at least three independent blots were measured, and data is presented as the mean value of biological replicates +/- standard error of the mean. Statistical significance was measured via Student’s two tailed t-test.