Validation of BAF-specific antibodies in lamin-KO MEFs, the effect of BAF overexpression on accumulation kinetics of the LC mutants at the rupture sites. (A and B) Validation of anti-BANF1 (3F10-4G12) for immunofluorescence after different fixation methods. Representative immunofluorescence images of single confocal sections from WT, Lmna^−/−, Lmnb1^−/−, and Lmnb2^−/− MEFs fixed with 4% PFA only (A) or 4% PFA containing 0.1% Triton X-100 (B) and stained with the anti-BANF1, followed with Cy5-labeled anti-mouse IgG, and Hoechst 33342 for DNA. Bars: 20 μm. (C and D) The effect of BAF overexpression on accumulation kinetics of the LC mutants at the rupture sites. Halo-BAF (lower panels) with mEmerald-LC full-length, ∆417-422 (∆NLS) and ∆432-572 (∆Tail; all, upper panels) were expressed in Lmna-KO MEFs. (C) Dynamics of mEmerald-LC full-length, ∆417-422 (∆NLS), ∆432-572 (∆Tail), and Halo-BAF. The right four columns are magnified views of orange boxes, and the rupture sites are indicated with yellow arrowheads. Bars: 5 μm (the first column) and 2 μm (the second column to others). (D) Relative fluorescence intensities of the mEmerald-LC mutants (means ± SEM; n = 10 cells; **, P < 0.001 from full-length by a linear mixed model).