Arpp19 knockout perturbs the phosphorylation and localization of key nuclear envelope proteins. (A) GFP- or GFP-Cre–expressing adenoviruses were enriched in mitotic cells using nocodazole and collected for Western blotting (lower panel) or processed for immunostaining (upper left panel). The phosphorylated lamin A/C at S22 signal was quantified (upper right panel). (B) GFP or Arpp19^Δ/Δ MEFs were synchronized (Noco) or not (Inter) in mitosis, lysed, and processed for immunoprecipitation (IP) using the mAB414 antibody that recognizes NUP 358, 214, 153, and 52 (Hofemeister and O’Hare, 2008). IPs were evaluated by Western blotting, using the antibody against serine phosphorylation by CDK (to detect NUP phosphorylation) and mAb414 to measure NUP levels. (C) GFP- or GFP-Cre–transduced Arpp19^Lox/Lox MEFs were analyzed by confocal microscopy using the mAB414 and anti-lamin A/C antibodies. Bar, 10 µm. Lamin A/C and NUP mean intensities during mitosis progression were measured using ImageJ. A line was drawn across the DNA mass on the DAPI channel and transposed to the lamin A/C (green) and NUPs (red) channels using the region of interest plug-in. A plot profile was then constructed, and NUP intensity was measured in two points on the profile located outside and at the surface of the DNA mass. The mean values of these two points were then used to calculate the percentage of nuclear NUPs relative to the total intensity. Lamin A/C intensities were calculated as for the NUP signals except that, because of the diffuse nuclear distribution of Lamin A/C, intensities were measured outside and inside the DNA mass. Lamin A/C and NUP intensity data (mean ± SD) are from 57 GFP- and 59 GFP-Cre–transduced MEFs from three different experiments.