Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: marker for neuronal apoptosis.
Alpha II-spectrin (alpha-fodrin) is a demonstrated endogenous substrate for caspase-3 in neurons undergoing unscheduled apoptotic death. We have previously identified the caspase cleavage site that yields the distinctive 120 kDa spectrin breakdown product (SBDP120) as (DSLD(1478)*SVEAL). Here, by using a synthetic peptide (NH(2)-SVEALC) mimicking the neo-N-terminal of SBDP120 as antigen, we report the development of chicken antibodies that specifically recognize the SBDP120 generated by in vitro caspase-3 digestion of bovine alpha-spectrin on Western blot. These anti-SBDP120 antibodies recognize SBDP120 generated by two apoptotic challenges (staurosporine, EGTA) to human neuroblastoma SH-SY5Y cells. Yet they neither react with intact alpha-spectrin nor its other fragments on Western blots. These anti-SBDP120 work equally well in detecting SBDP120 generated in rat cerebellar granule neurons undergoing potassium withdrawal-induced apoptosis. In immunocytochemical studies, these antibodies also specifically stained apoptotic SH-SY5Y or CGN's undergoing apoptosis in a caspase- inhibitor-sensitive manner. These anti-SBDP120s might become powerful markers for apoptotic neurons in various neurological or neurodegenerative conditions in vivo.
Nath
R
R
Laboratory of Neuro-biochemistry, Department of Neuroscience Therapeutics, Ann Arbor, MI 48105, USA. rathna.nath@wl.com
Huggins
M
M
Glantz
S B
SB
Morrow
J S
JS
McGinnis
K
K
Nadimpalli
R
R
Wanga
K K
KK
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D017209:Apoptosis; D001665:Binding Sites; D015415:Biomarkers / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D002645:Chickens / Q000276:immunology; D006801:Humans; D008840:Microfilament Proteins / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D008970:Molecular Weight; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology*; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured; D013049:Spectrin
D000906:Antibodies; D015415:Biomarkers; D002352:Carrier Proteins; D053148:Caspase 3; D020169:Caspases; D008840:Microfilament Proteins; D010446:Peptide Fragments; D013049:Spectrin; C505407:CASP3 protein, human; C505410:Casp3 protein, rat