Figure 3. Fractionation approach to... | Rockefeller University Press

Figure 3.

$Figure 3. Fractionation approach to identifying factors limiting for nuclear growth. (A) X. laevis egg extract was subjected to high-speed centrifugation to separate the extract into cytosol (Cyto), light membrane (LM), and heavy membrane (HM). Stage 10 nuclei were incubated with the indicated fractions for 120 min. IBB, added at ∼30 µM, inhibits nuclear import (Weis et al., 1996). Nuclear CS areas were measured for at least 200 nuclei per condition and normalized to the preincubation nuclear size. Data from two independent experiments are shown. (B) Experiments were performed as in Fig. 1, C–E, except that extracts were supplemented with IBB or wheat germ agglutinin (WGA) to block nuclear import (Cox, 1992). For each experiment, 7–212 nuclei were quantified at each time point (53 nuclei on average). Data from three different extracts are shown. The control data are the same shown in Fig. 1 E. (C) Schematic diagram of the fractionation approach. See Materials and methods for details. (D) Mono Q fractions were dialyzed into XB and concentrated ∼10- to 20-fold. Stage 10 embryo extract and nuclei were supplemented with equivalent volumes of XB or Mono Q fractions and incubated for 90 min. Nuclei were fixed and stained with NPC antibody mAb414. Nuclear CS areas were measured and the fold change was calculated relative to the preincubation nuclear size. Means and SDs from three independent fractionation experiments are shown. At least 160 nuclei were quantified for each condition. The dotted line indicates the top of the error bar for the XB control. We selected fractions with the largest fold changes in nuclear size and with SD error bars above the dotted line, namely, fractions 2 and 9, which were shown by mass spectrometry to contain Npm2. Error bars represent SD.$

Fractionation approach to identifying factors limiting for nuclear growth. (A) X. laevis egg extract was subjected to high-speed centrifugation to separate the extract into cytosol (Cyto), light membrane (LM), and heavy membrane (HM). Stage 10 nuclei were incubated with the indicated fractions for 120 min. IBB, added at ∼30 µM, inhibits nuclear import (Weis et al., 1996). Nuclear CS areas were measured for at least 200 nuclei per condition and normalized to the preincubation nuclear size. Data from two independent experiments are shown. (B) Experiments were performed as in Fig. 1, C–E, except that extracts were supplemented with IBB or wheat germ agglutinin (WGA) to block nuclear import (Cox, 1992). For each experiment, 7–212 nuclei were quantified at each time point (53 nuclei on average). Data from three different extracts are shown. The control data are the same shown in Fig. 1 E. (C) Schematic diagram of the fractionation approach. See Materials and methods for details. (D) Mono Q fractions were dialyzed into XB and concentrated ∼10- to 20-fold. Stage 10 embryo extract and nuclei were supplemented with equivalent volumes of XB or Mono Q fractions and incubated for 90 min. Nuclei were fixed and stained with NPC antibody mAb414. Nuclear CS areas were measured and the fold change was calculated relative to the preincubation nuclear size. Means and SDs from three independent fractionation experiments are shown. At least 160 nuclei were quantified for each condition. The dotted line indicates the top of the error bar for the XB control. We selected fractions with the largest fold changes in nuclear size and with SD error bars above the dotted line, namely, fractions 2 and 9, which were shown by mass spectrometry to contain Npm2. Error bars represent SD.