Immunolocalization of detergent-susceptible nucleoplasmic lamin A/C foci by a novel monoclonal antibody.
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 C3H10T(1/2) 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, C3H10T(1/2) cells were serum-starved and then refed to trigger cells to enter the G(1) 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.
Muralikrishna
Bh
B
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007, India.
Thanumalayan
S
S
Jagatheesan
G
G
Rangaraj
Nandini
N
Karande
Anjali A
AA
Parnaik
Veena K
VK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology*; D018604:Epitope Mapping; D016193:G1 Phase; D006801:Humans; D034904:Lamin Type A / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D051381:Rats
D000911:Antibodies, Monoclonal; D003902:Detergents; D034904:Lamin Type A; D009687:Nuclear Proteins; C440308:lamin C