Figure 3. Emerin phosphorylation on Y74 and Y95 mediates the mechanical adaptation of isolated nuclei to force.

a, Nuclei isolated from Hela cells were incubated with anti nesprin-1-coated magnetic beads and stimulated with a permanent magnet for 3 min. Tyrosine phosphorylation of nuclear proteins was analyzed by western blot. All results are representative of at least three independent experiments.

b, Nuclei isolated from emerin knockdown Hela cells re-expressing WT, Y59F, Y74F, Y95F or 74-95FF emerin mutants were incubated with anti nesprin-1-coated magnetic beads and stimulated with a permanent magnet for 3 min. Tyrosine phosphorylation of emerin mutants was analyzed by western blot after immunoprecipitation ("total" refers to the emerin level in nuclear lysates). Corresponding densitometric analysis (lower panel) of emerin phosphorylation normalized to emerin levels and expressed as relative to the control in the absence of stimulation by force (Error bars represent s.e.m, densitometric data were analyzed from n=4 independent experiments).

c, Change in bead displacement between the first and 6th pulse of force applied to beads coated with anti nesprin-1 antibody bound to nuclei isolated from emerin knockdown cells re-expressing WT (n=15 beads) or 74-95FF emerin mutants (n=18 beads). Displacements were calculated relative to the first pulse of force applied to nuclei isolated from emerin knockdown cells (Error bars represent s.e.m., *P<0.05, data were collected from 3 independent experiments and analyzed by two-tailed unpaired t-test).

d, Nuclei isolated from emerin knockdown Hela cells re-expressing WT or 74-95FF emerin mutants were incubated with anti nesprin-1-coated magnetic beads and stimulated with a permanent magnet for 3 min. After stimulation the nuclei were lysed with detergent (1% NP-40 in Tris buffer). Then, the protein complexes associated with the beads (bead complex) were isolated from the lysate using a magnetic separation stand and both fractions were denatured and reduced in Laemmli buffer. All results are representative of at least three independent experiments.

e, Emerin tyrosine phosphorylation was analyzed after immunoprecipitation in MRC5 cells cultured on matrix with different rigidity (polyacrylamide gels of 1 kPa and 50 kPa and plastic) and treated with blebbistatin. ("total" refers to the emerin level in nuclear lysates). Corresponding densitometric analysis (left panel) of emerin phosphorylation normalized to emerin levels and expressed as relative to the 1 kPa condition (Error bars represent s.e.m., densitometric data were analyzed from n=4 independent experiments).