p.R388P mutation alters solubility and nuclear envelope targeting of lamin A.

<p><b>A)</b> Whole cell extracts of C2C12 cells overexpressing wild-type (WT), R388P (RP), L647R (LR) FLAG-LA or WT FLAG-mature LA (mLA) were analysed by western blot using anti-FLAG, anti-LA/C (which recognise ectopic and endogenous A-type lamins), anti-prelamin A (preLA) and anti-GAPDH antibodies. <b>B)</b> C2C12 cells overexpressing WT or R388P FLAG-LA were fixed, labelled with anti-FLAG antibodies, and immunofluorescence observed at the confocal microscope. Representative cells presenting either a strong FLAG-lamin A staining at the nuclear envelope (NE), a predominant nucleoplasmic staining (Nu.) or the presence of small aggregates with ring—like structures (Ag.) are shown. Scale bar, 10 μm. <b>C)</b> Percentage of the three phenotypes NE, Nu. and Ag. for cells expressing WT or R388P FLAG-LA, as indicated. Data are the mean ± s.e.m. of 5 independent experiments and 100–250 nuclei/condition, *** p < 0.001 (two-way ANOVA with Bonferroni post-tests). <b>D)</b> C2C12 cells overexpressing WT or R388P FLAG-LA underwent cell fractionation to enrich the insoluble nuclear matrix intermediate filament scaffold. Proteins from soluble (S1, S2, S3) and insoluble (Ins.) fractions as well as whole cell extracts (T) were analysed in parallel by western blot using anti-FLAG antibodies. Samples loaded in each lane correspond to an identical cell number. Red Ponceau staining of nitrocellulose membrane illustrates the protein specificity of each fraction. <b>E)</b> ECL signals obtained in <b>D)</b> were scanned and quantified. The graph illustrates the distribution of FLAG tagged proteins into the different fractions assuming that 100% of the signal correspond to the total of signals recovered in [S1 + S2 + S3 + Ins.]. Data are the mean ± s.e.m. of 3 independent experiments. ** p < 0.01 and * p < 0.05 (two-way ANOVA with Bonferroni post-tests).