Immunolocalization of detergent-susceptible nucleoplasmic lamin A/C foci by a novel monoclonal antibody

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Abstract
The A‐type lamins are localized in the interior of the nucleus as well as on the nuclear periphery. In this study, we have characterized a monoclonal antibody LA‐2F9 produced against recombinant rat lamin A which stains a subpopulation of various cell types in a pattern of small nucleoplasmic foci that are unusually susceptible to mild detergent/salt extraction. The specific reactivity of mAb LA‐2F9 towards lamins was confirmed by immunoblotting of HeLa and C3H10T½ whole cell lysates and nuclear lysates. The epitope for LA‐2F9 was narrowed down to amino acid residues 268–278 (SAKLDNARQSA). To check whether the appearance of lamin foci was cell‐cycle‐dependent, C3H10T½ cells were serum‐starved and then refed to trigger cells to enter the G1 phase of the cell‐cycle. The intensity of staining increased 3.5‐fold within 6 h of refeeding, when the maximum number of cells were labeled with LA‐2F9. We also checked whether the LA‐2F9 foci colocalized with nuclear proteins known to be distributed in small foci such as hnRNPs, snRNPs, SC‐35, and p80 coilin, but did not find evidence of colocalization. Our studies suggest that LA‐2F9 has a novel and specific reactivity towards detergent‐susceptible lower order lamin structures that are likely to be assembly intermediates.