Figure 1.

Illustration of cytomatrix isolation

The cytosol and cytomatrix were sequentially extracted from HCT-15 cells. The cytosol was extracted with nonionic detergent containing mild solvent, whereas the cytomatrix was separated from the nuclear fraction using a buffered, stringent solvent.

(A) Immunoblots of marker proteins in fractions.

(B) Immunofluorescence image of keratin localization in cytosol-removed cells.

(C) Immunofluorescence images of SUN2 (red) and Nesprin-1 (green) localization in HeLa cells.

(D) Nesprin-1 is predominantly localized around the nucleus in SUN2 knockdown cells.

(E-J) TEM micrographs of HCT-15 cells. Ultrastructure of the nuclear periphery of (E and F) an intact cell and (G-I) a cytosol-removed cell. (J) cytosol and cytomatrix removed core nuclei.

(K) Protein expression in the cytosol versus cytomatrix of three cell lines. The cytosol and cytomatrix were run side by side, but bands were cut based on cell line to show differences in protein expression in corresponding compartments. Western blots were run in the same gel. The three cell lines show consistent results for predominantly cytosolic proteins (upper panel), equally distributed proteins (middle panel), and predominantly cytomatrix proteins (bottom panel). Cyt – cytosol, CMX – cytomatrix, M− mitochondria, INM – inner nuclear membrane, ONM – outer nuclear membranes, PM – plasma membrane carcass, PNS – perinuclear space.