Stain free gel

In vitro phosphorylation of Rif1–CTD by Plk1 was performed by adding 590 nM recombinant Plk1, 0.1 μCi [γ³²P]-ATP (Hartmann Analytic, Braunschweig, Germany), 50 μM ATP, 6 μM of recombinant Rif1–CTD or Casein as control substrate for 30 min at 23°C in kinase buffer (20 mM HEPES, pH 7.2, 10 mM MgCl_2, 2 mM DTT, 0.1 mM EGTA) with or without 100 nM Plk1 inhibitor BI2536. Reactions were stopped in Laemmli sample buffer, proteins were separated by SDS-PAGE. Gels were dried and bands were quantified on a Phosphorimager Typhoon Trio (GE Healthcare). An aliquot of each reaction was withdrawn before the addition of [γ³²P]-ATP and loaded on a Stain-Free gel (BioRad) for visualization. For phosphorylation assay in extracts, Rif1–CTD (1 μM) was incubated in mock or Plk1 depleted egg extract for 1 h in the presence of 0.1 μCi [γ³²P]-ATP and energy mix, at 22°C. Reactions were stopped by dilution in three volume of slurry Nickel Sepharose beads in EB, binding was allowed for 20 min under shaking at 4°C, beads were washed twice with EB/NP-40 (0.1%) and four times in EB, bound proteins were eluted by Laemmli sample buffer, followed by 12% SDS-PAGE and quantification. Rif1–CTD pulldown assays for PP1 binding were performed using magnetic Ni-NTA beads (CubeBiotech, Monheim, Germany).

Cultures were grown overnight shaking at 37°C with appropriate antibiotics. The next day, the cultures were centrifuged and the pellets were separated from the supernatants. The pellets were resuspended in lysis buffer (50 mM Bis-Tris pH 7.5, 4% SDS, 0.1 mg/mL lysozyme, 25 U benzonaze), and then lysed by bead beating (FastPrep system, MP Biomedicals). Proteins were precipitated from the supernatants by methanol-chloroform precipitation and resuspended in water. Proteins from the pellets and supernatants were quantified by BCA analysis (ThermoFischer), run on a Stain-Free gel (BioRad). Total protein was visualized by Stain-Free^™ imaging technology using a Bio-Rad ChemiDoc MP imager. Protein bands were then transferred to a nitrocellulose membrane by semi-dry transfer. The membrane was blocked for 1 hour with TBST+5% powdered milk at room temperature, then stained with primary antibody (rabbit anti-SagA polyclonal sera) diluted 1:50,000 in TBST+5% milk for 1 hour at room temperature. The membrane was then stained with secondary antibody (goat anti-rabbit antibody, HRP-conjugate) diluted 1:20,000 in TBST+5% powdered milk for 1 h at room temperature with agitation. Membranes were washed with TBST 3 times for 5 min at room temperature with agitation. Blots were developed using Clarity Western ECL substrate (Bio-Rad) and imaged using a Bio-Rad ChemiDoc MP imager.