Dmrbe dmire2

Cells were fixed with 4% PFA/PBS for 20 min at room temperature, with or without methanol post-fixation ( -20°C for 20 min). Samples were blocked with 1% BSA/PBS, stained with the indicated antibodies and counterstained with ToPro3/DAPI (Jackson ImmunoResearch Laboratories). Co-localisation was quantitated by detecting overlapping pixels with Imaris v8.0 (Bitplane) and analysed with Pearson’s Correlation Coefficient [24 (link)]. For PLA, PFA fixed cells were subjected to the Duolink Proximity Ligation Assay (Sigma) as described by the manufacturer. Confocal microscopy was performed on Leica DMRBE/DMIRE2. Images were analysed with Imaris where individual spots were defined with a variable and initial size estimate of 0.5 μm. Images were processed with Adobe Photoshop, and adjusted for optimal brightness/contrast. Minimal gamma changes were made to enable visualisation of overlaid signals.

PLA validation data in Fig. 3 was performed in fixed cells using antibodies to CDK2 and Cyclin E1 with detailed methodology described in^{37 (link)}. Cells were fixed with 4% PFA/PBS for 20 minutes at room temperature, with or without methanol post-fixation (−20 °C for 20 min). Samples were blocked with 1% BSA/PBS, stained with the indicated antibodies and counterstained with ToPro3/DAPI (Jackson ImmunoResearch Laboratories). PFA fixed cells were subjected to the Duolink Proximity Ligation Assay (Sigma) as described by the manufacturer using antibodies CDK2 (M2, D12) and cyclin E1 (HE12) (Santa Cruz Biotechnology). Confocal microscopy was performed on Leica DMRBE/DMIRE2. Images were analysed with Imaris where individual spots were defined with a variable and initial size estimate of 0.5 μm. 7 composite (foci, nuclear and cytoplasmic) images were used for image analysis. Images were processed with Adobe Photoshop, and adjusted for optimal brightness/contrast. Minimal gamma changes were made to enable visualisation of overlaid signals. A table of the optimised image parameters used for the PLA image analysis is provided in Supp. Table 6.