Figure 1. Barrier-to-autointegration factor nuclear localization in prelamin A-accumulating diseases. (A) BAF and prelamin A distribution in control (cont.1 adult healthy donor, cont.2 neonatal healthy donor) Familial partial lipodystrophy (FPLD, LMNA-R482Q), Mandibuloacral dysplasia (MADA, LMNA-R527H) and Restrictive dermopathy (RD, ZMPSTE24 c.1085_1086InsT). Control and pathological cells at early passage (passage 5–10) were used for each experiment. BAF was evaluated on methanol-fixed cells by a rabbit-polyclonal anti-BAF antibody visualized by FITC-conjugated secondary antibody (green). Prelamin A was evaluated using a goat-polyclonal anti-prelamin A antibody visualized by TRITC-conjugated secondary antibody (red). In merge, arrow, arrowhead and asterisk indicate double-stained nuclei. DNA was detected using DAPI. Bar, 10 μm. (B) Higher magnification of prelamin A-BAF-positive nuclei indicated by arrow, arrowhead and asterisk in panel A. Prelamin A (red), BAF (green) merge (merge) and merge plus DAPI staining (merge + DAPI) are shown. (C) The percentage of nuclei showing prelamin A staining is reported in the graph as means ± SD of three different counts (100 nuclei per count). Examined samples are control fibroblast from adult healthy donors (cont.1), familial partial lipodystrophy fibroblasts (FPLD), mandibuloacral dysplasia fibroblasts (MADA), control fibroblast from neonate healthy donors (cont.2) and restrictive dermopathy fibroblasts (RD). Asterisks indicate statistically significant differences at the Student’s t-test (p < 0.05), with respect to control cells values. (D) Western blotting evaluation of BAF protein levels in control (cont.1 and cont.2), FPLD, MADA and RD cells. Eighty micrograms of total cell lysates were subjected to western blot detection of prelamin A (prelamin A), lamin A/C (lamin A/C), BAF (BAF) and actin (actin). Immunolabeled bands visualized by ECL chemiluminescence are shown.