Figure 3.

Localized prelamin A detection around the nuclear periphery via immunohistochemical analysis using the PL-1C7 antibody. (A) and (B). Two examples of Dox-treated GFP-Lmna MEF cells stained with prelamin A antibody PL-1C7 (red) and counter-stained with DAPI. GFP signal represents preferentially the mature lamin A, but also the prelamin A fraction due to the GFP tag in the N-terminus (Fig. 2). Dotted boxes show regions where correlation analyses between GFP-lamin A and GFP-prelamin A were performed (dotted line). Signal intensities were normalized to the highest value (100). The signal distribution pattern of the GFP-fusion proteins (GFP signal) representing primarily mature lamin A/C (green line; GFP fusion) is significantly different from the prelamin A distribution pattern detected by the PL-1C7 antibody (red line; prelamin A). (C) and (D). Immunostaining for lamin B, lamin A/C and prelamin A (PL-1C7) in wild-type C2C12 cells. Lamin A/C and prelamin A distribution around the nuclear periphery was analyzed as described in (A) and (B). Scale bar: 5 μm. (E). Simulated measurements to show the utility of the ‘fluctuation index’ metric to assess differences in spatial localization of a target protein relative to a reference protein. The fluctuation index increases as the TEST localization pattern becomes increasingly punctate (Case 1, 2 and 3) relative to a reference pattern. (F). Lamin A/C /lamin B fluctuation index in immunostained C2C12 nucleus (n = 44). (G). Prelamin A/lamin B fluctuation index in immunostained C2C12 nucleus (n = 44).