Monoclonal antibody against microtubule associated protein-1 produces immunofluorescent spots in the nucleus and centrosome of cultured mammalian cells.

Abstract

ABSTRACT.A monoclonal antibody was raised against the highest molecular weight protein associated with microtubules (MAP-1). Its specific binding to MAP-1 was determined by immunoblotting of the gel electrophoretogram of microtubule proteins prepared from porcine brain. The antibody reacted only with MAP-1, not with MAP-2, tau or tubulin. Indirect Immunofluorescent staining by this antibody showed bright intranuclear spots, the centrosome and the faint meshwork of the cytoplasm in several types of cultured mammalian cells; HeLa, PtK2, human skin fibroblasts, mouse melanoma cells, Chinese hamster ovary cells. The nulcear spots in the interphase cells, were replaced by diffuse enhanced fluorescence throughout the cell except for chromosomes during mitosis. They reappeared in late telophase, first in the cytoplasm, late in the nucleus. The punctate pattern of nuclear immunofluorescence was not affected by microtubule-depolymerizing agents. The result that it persisted on residual cell structures after extraction with a high salt concentration buffer containing Triton X-100 followed by digestion with DNase I and RNase A suggests that the antigen is associated with the nuclear skeleton.

Microtubule-associated proteins (MAPs): a monoclonal antibody to MAP 1 decorates microtubules in vitro but stains stress fibers and not microtubules in vivo.

“…In addition to staining stress fibers, mAb 7-1.1 stained interphase nuclei, often in a speckled or punctate fashion (Fig. 4), as described for MAP 1 antibody staining by other investigators (11). The number of speckles observed in mouse (Fig.…”

A monoclonal antibody to chicken gizzard desmin that recognizes intermediate filaments and nuclear granules in BHK21/C13 cells.

“…Some, including the anti-MAP-1 MAbs (2,8,45,46) and the MAbs to 36 kD and 40 kD nuclear matrix protein (36), however, differ from AC54 MAb in the staining pattern of the nucleus; they stained nuclear spots that were not located at the nucleolus, whereas AC54 MAb stained nuclear granules that sometimes were concentrated at the nucleolus (Fig. 2 f).…”

“…These include anti-MAP (microtubule associated protein)-1 MAbs (2,8,45,46); MAb to 240 kD MAP (42); MAb to a 52 kD nuclear matrix protein (14); MAbs to 36 kD and 40 kD nuclear matrix proteins (36); MAbs to non-lamin nuclear matrix antigens (9); CNMI-21 MAb to a nuclear lysate (58); B100 MAb to an IF-associated protein, p50 (63); S-30 MAb to a 57 kD protein present only in non-proliferating or senescent cells (62); antibodies to nuclear thyroid hormone-binding proteins (47); anti-PCNA (proliferating cell nuclear antigen) sera from patients with systemic lupus erythematosus (55); anti-SS-B sera from patients with Sjogren's syndrome (12); and anti-protein BA antibody that reacts with a non-histone protein specific to normal, non-proliferating tissues (3).…”

Co-localization of SV40 T antigen and p53 with immunological analogues of microtubule-associated protein-1 on the nuclear skeleton.

“…Antiserum against MAP-1 produced immunofluorescence on the centrosome (4), and a monoclonal antibody against MAP-1A stained the mitotic spindle, cytoplasmic microtubules and spots on the nucleus (5). Our monoclonal antibody against MAP-1 showed specific binding to both of the major bands of MAP-1 on immunoblotting a gel electrophoretogram of microtubule proteins prepared from porcine brain (6). Indirect immunofluorescent staining by this antibody made visible bright intranuclear spots bound to the nuclear skeleton, centrosomes and a faint network of the cytoplasm in several types of mammalian cells (6).…”

“…Our monoclonal antibody against MAP-1 showed specific binding to both of the major bands of MAP-1 on immunoblotting a gel electrophoretogram of microtubule proteins prepared from porcine brain (6). Indirect immunofluorescent staining by this antibody made visible bright intranuclear spots bound to the nuclear skeleton, centrosomes and a faint network of the cytoplasm in several types of mammalian cells (6). Benett reported the resemblance of MAP-1 to erythrocyte ankyrin in its immunofluorescent staining of microtubules and nuclear spots and in their immunoblotting (7).…”