Xenbase Image ID: 146824

FIGURE 1. Lamin B3 concentration affects nuclear size in X. laevis egg extract. A) Nuclei were pre- assembled in X. laevis egg extract for 30-35 minutes. Recombinant GFP-LB3 was added to the indicated final concentrations, and the same total volume was added to each reaction. For the control reaction, an equal volume of lamin storage buffer was added. Nuclear growth was allowed to proceed for an additional 75 minutes at 20°C. Nuclei were fixed, spun onto coverslips, processed for immunofluorescence, and stained with mAb414 to visualize the NPC (red) and Hoechst to visualize the DNA (blue). Representative images are shown. White arrows denote intranuclear lamin puncta that form upon addition of high concentrations of recombinant lamin proteins. B) For the representative images at the top of the panel, immunofluorescence against GFP was performed, demonstrating that low concentrations of GFP-LB3 properly localize to the NE. The bar graph shows nuclear size data from one representative experiment. For each bar, the cross-sectional areas of 150-870 nuclei were measured from NPC-stained nuclei and averaged. The error bars are SD. * p<0.05, ** p<0.01, *** p<0.001, NS Not significant. C) Cumulative data from 20 different X. laevis egg extracts are shown. For each data point, the average cross-sectional nuclear area was measured as in B) and normalized to the buffer addition control (bold horizontal line set at 1.0). The red symbols represent data for LB3 protein without a GFP tag. The error bars are SEM. All normalized nuclear area data points greater than 1.05 and less than 0.94 are statistically different from the buffer control (1.0) by at least p<0.05. Due to variability between egg extracts, the data were normalized to the buffer addition control, and SEM error bars are shown rather than larger SD error bars that obscure visualization and interpretation of the data. D) Similar experiments were performed except that mutant LB3 proteins were added at low (2 nM) or high (34 nM) concentrations. The blue data are for LB3 without a GFP tag. The red data are for GFP-LB3 in which the CAAX box was mutated so it cannot be farnesylated (LB3 amino acids 578 and 579 mutated from cysteines to serines). The green data are for GFP-LB3 with an R385P point mutation within LB3 that renders the protein defective for assembly into the nuclear lamina. For each bar, the cross-sectional areas of 152-944 nuclei were measured from NPC-stained nuclei, averaged, and normalized to the buffer addition control (set at 1.0). The error bars are SEM. * p<0.05, ** p<0.01, *** p<0.001, NS Not significant. Image published in: Jevtić P et al. (2015) Copyright © 2015. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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