An antigen cross-reacting with anti-laminin sera is found in the submembranous cortical region of various cells in culture.
Laminin is a complex extracellular matrix molecule consisting of one A-subunit (Mr400KD) and 3 B-subunits (Mr220KD) and is found in the basement membrane. Even though it is now apparent that different cell types are synthesizing laminin-like molecules, the role of these molecules in different systems is not well understood. We have characterized laminin and raised specific antiserum in rabbits. The distribution of laminin was studied by indirect immunofluorescence in different cells such as PFHR-9, WI-38, MRC-5, CHO, 3T3, WI38VA132RA, RAW264-7 and Ki3T3. All normal and transformed cells display a high amount of intracellular submembranous network-like component cross-reacting with antilaminin serum (anti-Lm) and not with anti-fibronectin (anti-Fn) serum as seen by immunofluorescence in permeabilized cells. Preabsorption of anti-Lm with increasing amounts of laminin progressively decreased the staining of the submembranous network. Anti-Lm sera from four other laboratories also showed similar staining pattern. The structural and non-secretory nature of this submembranous staining was confirmed by (a) inhibiting protein synthesis in 0.5% serum and 4 micrograms/ml puromycin and (b) by immunoelectron microscopy of permeabilized cells. Immunoprecipitation of 3H-leucine labelled cellular proteins with anti-laminin sera showed proteins of Mr 220-210 KD in SDS-PAGE fluorography. These studies suggest that an antigen(s) crossreacting with anti-Lm sera is localized in the membrane associated cytoskeletal region where spectrin/fodrin family of proteins have been localized.
D000818:Animals; D000941:Antigens / Q000032:analysis*; D001485:Basement Membrane / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis*; D002467:Cell Nucleus / Q000032:analysis; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000032:analysis*; D005109:Extracellular Matrix / Q000032:analysis; D005353:Fibronectins / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007163:Immunosorbent Techniques; D007797:Laminin / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008854:Microscopy, Electron; D008985:Monensin / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology
D000941:Antigens; D005353:Fibronectins; D007106:Immune Sera; D007797:Laminin; D011691:Puromycin; D008985:Monensin; D003513:Cycloheximide