For gel-shift Western blots, whole-cell extracts were lysed in 100 µl of 3× Western blot sample buffer in a Mini-beadbeater-16 (Biospec) for 2 min. Gels were run at a constant 20 mAmps until a 75-kD marker was at the bottom of the gel. Blots were probed with mouse anti-FLAG M2 (Sigma-Aldrich) and rabbit anti-Wee1 (Deng and Moseley, 2013 (link)). For Wee1 Western blots, we also repeated each experiment with a different rabbit anti-Wee1 antibody (provided by D.R. Kellogg; Lucena et al., 2017 (link)), which yielded identical results (not depicted). For monitoring Wee1 phosphorylation, samples were run on an SDS-PAGE gel containing 6% acrylamide and 0.02% bisacrylamide.
For sucrose-gradient Western blots, samples were prepared as described above and run at a constant voltage of 100 mV on 10% acrylamide gels. Blots were probed with mouse anti–FLAG M2, rabbit anti–Cdr2-pT166 (Deng et al., 2014 (link)), or mouse anti-HA (clone 16B12; BioLegend). To reduce background signal, nitrocellulose membranes for phosphospecific Western blots were incubated in 4% BSA/TBS–Tween-20 for both primary and secondary antibodies at 4°C with 2-h washes in between.
For sucrose-gradient Western blots, samples were prepared as described above and run at a constant voltage of 100 mV on 10% acrylamide gels. Blots were probed with mouse anti–FLAG M2, rabbit anti–Cdr2-pT166 (Deng et al., 2014 (link)), or mouse anti-HA (clone 16B12; BioLegend). To reduce background signal, nitrocellulose membranes for phosphospecific Western blots were incubated in 4% BSA/TBS–Tween-20 for both primary and secondary antibodies at 4°C with 2-h washes in between.
