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FIGURE 3. Lamin combinations affect nuclear size similarly to single lamins in X. laevis egg extract. A) Western blots were performed to estimate the endogenous amounts of different lamins present in X. laevis embryos during development. Known amounts of recombinant lamins were loaded to the left side of each gel (GFP-LB1: 0.45, 0.9, 1.8 pmol; GFP-LB2: 0.5, 1.25, 2.5 pmol; GFP-LA: 0.1, 0.15, 0.2 pmol). 10 μl each of different stage X. laevis embryo extracts were loaded to the right side of each gel. Blots were probed with antibodies previously shown to be specific to each lamin type (data not shown), as well as Ran as a loading control. Absolute protein amounts were determined by comparing band intensities, quantified by infrared fluorescence, to the known amounts of recombinant lamins on the same blot. Embryo extracts prepared from three different frogs were quantified, and one representative western blot is shown for each lamin. Values below each lane represent the mean ± SD (n=3). LB3 amounts were similarly measured previously as 0.75 pmol in the egg, 2.6 pmol in stage 8, 0.3 pmol in stage 11, and negligible in later stages (6). The bar graph shows average total lamin concentrations present at each stage of development with SD error bars (n=3). B) Similar experiments were performed as in Figure 1, except that different lamin combinations were added to nuclei pre-assembled in egg extract (see the Figure Legend for Figure 1). Lamin combinations that approximated stage 8 were: 175 nM LB3 and 50 nM LB1 (blue diamonds). Lamin combinations that approximated stage 11 were: 50 nM LB1 and 50 nM LB2 (red squares). Lamin combinations that approximated stage 19 were: 20 nM LB1, 20 nM LB2, and 5 nM LA (green triangles). Cumulative data from 5 different X. laevis egg extracts are shown. For each data point, the cross-sectional areas of 85-840 nuclei were measured from NPC-stained nuclei, averaged, and normalized to the buffer addition control (bold horizontal line set at 1.0). The error bars are SEM. All normalized nuclear area data points are statistically different from the buffer control (1.0) by at least p<0.01. 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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