Characterization of two DNase gamma-specific monoclonal antibodies and the in situ detection of DNase gamma in the nuclei of apoptotic rat thymocytes.
Two novel monoclonal antibodies (mAbs), hg302 and hg303, raised against a synthetic peptide corresponding to the basic domain of human DNase gamma, are characterized in detail. In Western blot analysis, hg303 recognizes both wild type and C-terminal Myc-His-tagged human DNase gamma, but does not cross-react with human DNase I family members, DNase I, DNase X, or DNAS1L2. On the other hand, dot blot analysis reveals the fine specificity of hg302; it recognizes human, mouse, and rat DNase gamma, but not other DNase I family DNases under non-denaturing conditions. Furthermore, hg302 efficiently immunoprecipitates wild type, but not C-terminal Myc-His-tagged, human DNase gamma from cell lysates. In immunohistochemical analysis, hg302 strongly recognizes DNase gamma in the nuclei of X-ray irradiation-induced apoptotic, but not normal rat thymocytes. The specific detection of DNase gamma in apoptotic nuclei is confirmed by indirect-immunofluorescence analysis using TNF-alpha-induced apoptotic HeLa S3 cells transfected with DNase gamma. These results, together with the observations that DNase gamma is present in normal thymocytes and its activity is unchanged during the apoptosis, suggest that some molecular change(s), which triggers the activation of DNase gamma, occurs in response to apoptotic stimuli in the basic domain, and hg302 specifically recognizes the activated DNase gamma in immunohistochemical analysis.
Shiokawa
D
D
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12 Funagawara-machi, Ichigaya, Shinjuku-ku, Tokyo, 162-0826, Japan.
Tanaka
M
M
Kimura
T
T
Hashizume
K
K
Takasawa
R
R
Ohyama
H
H
Fujita
K
K
Yamada
T
T
Tanuma
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D017209:Apoptosis; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D017931:DNA Primers; D004706:Endodeoxyribonucleases / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013950:Thymus Gland / Q000166:cytology / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004706:Endodeoxyribonucleases; C105124:deoxyribonuclease gamma