JCS019968 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Total membranes from Xenopus egg extracts were extracted with 100 mM Tris (pH 7.5), 1 M NaHCO₃, 7 M urea, 3M urea, 1% Triton X-100 plus 0.25 M NaCl, 1% Triton X-100 or 1 M KCl. Extracted membranes were divided into soluble (S) and pelletable (P) fractions, resolved by 10% SDS-PAGE and immunoblotted with CEL5C, LAP12, CEL13A or CEL1FF antibodies. Molecular masses (kDa) were calculated from scanned blots using UVIband map software.

• Supplemental Figure S2 -

  Fig. S2. Timing of association of NEP-A and NEP-B antigens with sperm pronuclei assembled in vitro. Sperm pronuclei were assembled in Xenopus egg extracts. At intervals of ten minutes, nuclei were fixed and stained with monoclonal antibody 4G12 (A), CEL1FF (B), CEL13A (C) or XLAP2 (D). All samples were counter stained with DAPI. In each panel, DAPI images and antibody staining are shown as individual mono micrographs (left-hand and central panels, respectively) or as two-colour merged images (right-hand panels). Bar, 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Deletion constructs of human emerin. (A) Deletion constructs of human emerin encoding residues 1-70 or 73-180 were generated. (B) The constructs were expressed in Escherichia coli and purified on nickel columns. Purified fractions were resolved by 12% SDS-PAGE and stained with Coomassie Blue (central panels) or immunoblotted with antibody against emerin (right-hand panels). Molecular mass markers are in the left-hand panels. Arrows show the position of bacterial heatshock proteins. (C) The ability of each construct to bind to condensed (upper panels) or decondensed (lower panels) chromatin was tested by incubating the constructs with the relevant sperm chromatin templates and then performing immunofluorescence with antibodies against emerin followed by FITC-labelled goat anti-mouse antibody. DNA was detected with DAPI. Bar, 5 µm.