Planapo 60x 1.4 n a oil immersion objective

Images to quantify relative fluorescence intensities within nuclei were acquired using the above described spinning disk confocal system with an Olympus PlanApo 60x/1.4 N.A. oil-immersion objective heated to 37 °C, and Volocity software (PerkinElmer). Single Z-slices of nuclei were acquired using the 568 nm laser at 400 ms exposure and 15% laser power. Nuclei were segmented manually, and mean fluorescence calculated and normalised to that of RING1B and the number of RING1B molecules determined as above. Calculated numbers of molecules are given in Supplementary Table 4.

Wild type and RING1B-HaloTag cells were passaged together in a 1:1 ratio. Cells were trypsinised and labelled with Halo-TMR in suspension as described for fluorescence-based protein quantification above. Labelled cells were fixed with 3.3% formaldehyde for 10 min at room temperature. Permeabilisation (0.5% Triton X-100 in PBS), blocking (3% BSA in PBS for 30 min), primary (2.5 h, anti-RING1B, Cell Signalling Technology) and secondary antibody incubations (1 h, Supplementary Table 3) and DAPI incubation (0.1 μg/ml, 10 min) were all performed in suspension. Following all treatments, cells were resuspended in VECTASHIELD mounting medium (Vector Laboratories), and 10 μl of cells were flattened on a slide by applying pressure to a coverslip. Single Z-slices of nuclei were acquired using the above described spinning disk confocal system with an Olympus PlanApo 60x/1.4 N.A. oil-immersion objective and Volocity software (PerkinElmer). Signal from Halo-TMR in the 561 nm channel was used to differentiate wild type and RING1B-HaloTag cells. Signal from secondary antibody was acquired using the 488 nm laser at 10% laser power with 500 ms exposures. Nuclei were segmented manually and mean 488 nm channel fluorescence calculated for each cell line.