Western blot analysis of various cell lines, untreated (-) or treated with Staurosporine #9953 (1 μM; 3 hr) or with Etoposide #2200 (25 μM, overnight), using Caspase-3 (D3R6Y) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). MCF7 cells are negative for caspase-3 expression.
Western blot analysis of extracts from NIH/3T3 cells, untreated or staurosporine-treated (1 µM) and Jurkat cells, untreated or etoposide-treated (25 µM), using Caspase-6 Antibody.
Western blot analysis of extracts from Jurkat and A20 cells, untreated (-) or treated with Etoposide #2200 (25 μM, overnight; +), using Caspase-7 (D2Q3L) Rabbit mAb.
Western blot analysis of extracts from control HeLa cells (lane 1) or Caspase-8 knockout HeLa cells (lane 2) using Caspase-8 (1C12) Mouse mAb #9746 (upper), or β-actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the Caspase-8-knockout HeLa cells confirms specificity of the antibody for Caspase-8.
Western blot analysis of extracts from Jurkat cells (human), L929 cells (mouse), and C6 cells (rat), untreated or treated with staurosporine or cytochrome c as indicated, using Caspase 9 (C9) Mouse mAb.
Flow cytometric analysis of HeLa cells (green) using Lamin A/C (4C11) Mouse mAb (solid lines) or a concentration matched Mouse (G3A1) mAb IgG Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
Western blot analysis of extracts from control HeLa cells (lane 1) or HeLa cells with an apparent in-frame truncation mutation in the gene encoding LMNA (lane 2) using Lamin A/C (4C11) Mouse mAb #4777 (upper) or α-actinin (D6F6) XP® Rabbit mAb #6487 (lower). The change in LMNA molecular weight in the mutated HeLa cells is consistent with an in-frame deletion.
Western blot analysis of extracts from NIH/3T3 cells, untreated or staurosporine-treated (1 µM), and Jurkat cells, untreated or etoposide-treated (25 µM), using PARP Antibody.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from HCT116 cells (lane 1) or CASP3 knock-out cells (lane 2) using Caspase-3 (D3R6Y) Rabbit mAb #14220 (upper), and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). The absence of signal in the CASP3 knock-out HCT116 cells confirms specificity of the antibody for CASP3.
Western blot analysis of extracts from SKW6.4 cells, untreated or anti-Fas-treated (1 µg/ml), and Jurkat cells, untreated or etoposide-treated (25 µM), using Caspase-8 (1C12) Mouse mAb.
Immunofluorescent analysis of normal rat brain using Lamin A/C (4C11) Mouse mAb (green) and MAP2 Antibody #4542 (red).
Confocal immunofluorescent analysis of HeLa cells using Lamin A/C (4C11) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Lamin A/C (4C11) Mouse mAb.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Lamin A/C (4C11) Mouse mAb.
Western blot analysis of extracts from various cell lines using Lamin A/C (4C11) Mouse mAb.
Western blot analysis of extracts from THP-1 cells, untreated or treated with cycloheximide (CHX, 10 μg/ml, overnight) followed by TNF-α #8902 (20 ng/ml, 4 hr), using Lamin A/C (4C11) Mouse mAb.
|Caspase-3 (D3R6Y) Rabbit mAb 14220||20 µl||
||H M R Mk||35, 19, 17||Rabbit IgG|
|Caspase-6 Antibody 9762||20 µl||
||H M R||15, 35||Rabbit|
|Caspase-7 (D2Q3L) Rabbit mAb 12827||20 µl||
||H M R||20, 35||Rabbit IgG|
|Caspase-8 (1C12) Mouse mAb 9746||20 µl||
||H||18, 43, 57||Mouse IgG1|
|Caspase-9 (C9) Mouse mAb 9508||20 µl||
||H M R Hm Mk||47/37/35 (H). 49/39/37 (M). 51/40/38 (R).||Mouse IgG1|
|Lamin A/C (4C11) Mouse mAb 4777||20 µl||
||H M R Mk||74 (Lamin A), 63 (Lamin C)||Mouse IgG2a|
|PARP Antibody 9542||20 µl||
||H M R Mk||89, 116||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
|Anti-mouse IgG, HRP-linked Antibody 7076||100 µl||
The Procaspase Antibody Sampler Kit provides an economical means to evaluate the abundance and activation of caspases. The kit contains enough primary antibody to perform at least two western blots per primary antibody.
Each antibody in the Procaspase Antibody Sampler Kit detects endogenous levels of its respective target. Caspase-3 (D3R6Y) Rabbit mAb recognizes endogenous levels of total caspase-3 protein. This antibody detects full-length caspase-3 (35 kDa) as well as the large subunit (p20) of caspase-3 resulting from cleavage during apoptosis. Caspase-6 Antibody detects both full length caspase-6 (35 kDa) and the small subunit (15 kDa) of caspase-6 resulting from cleavage at Asp193. Caspase-7 (D2Q3L) Rabbit mAb detects both the full-length (35 kDa) and the large subunit (20 kDa) of caspase-7 resulting from cleavage at Asp198. Caspase-8 (1C12) Mouse mAb detects full length (57 kDa), the cleaved intermediate p43/p41, and the p18 fragment of caspase-8. Caspase-9 (C9) Antibody detects full-length caspase-9, as well as the large fragments resulting from cleavage at Asp315 and Asp330. PARP Antibody detects full length PARP1 (116 kDa), as well as the large fragment (89 kDa) of PARP1 resulting from caspase cleavage at Asp214. Lamin A/C (4C11) Mouse mAb detects full-length lamin A and lamin C proteins, as well as the larger fragments of lamin A (50 kDa) and lamin C (41 kDa) resulting from caspase cleavage. Caspase-9 (C9) Antibody detects the pro form of caspase-9 as well as cleaved fragments.
Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro158 of human caspase-7 protein, the carboxy-terminal sequence of the p18 fragment of human caspase-8 protein, recombinant human caspase-3 protein, caspase-9 protein, or human lamin A protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the cleavage site of caspase-6 or the caspase cleavage site in PARP. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Initiator caspases (including 2, 8, 9, 10 and 12) are closely coupled to proapoptotic signals, which include the FasL, TNF-α, and DNA damage. Once activated, these caspases cleave and activate downstream effector caspases (including 3, 6 and 7), which in turn cleave cytoskeletal and nuclear proteins like PARP, α-fodrin, DFF and lamin A, and induce apoptosis (1,2).
Caspase-8 (FLICE, Mch5, MACH) and Caspase-9 (ICE-LAP6, Mch6) are initiator caspases. CD95 receptor (Fas/APO-1) and tumor necrosis factor receptor 1 (TNFR1) activate caspase-8, leading to the release of the caspase-8 active fragments, p18 and p10 (3-6). Cytochrome c released from the mitochondria associates with procaspase-9 (47 kDa)/Apaf 1. Apaf-1 mediated activation of caspase-9 involves intrinsic proteolytic processing resulting in cleavage at Asp315 and producing a p35 subunit. Another cleavage occurs at Asp330 producing a p37 subunit that can serve to amplify the apoptotic response (7-11).
Caspase-3 (CPP-32, Apoptain, Yama, SCA-1), Caspase-6 (Mch2), and Caspase-7 (CMH-1, Mch3, ICE-LAP3) are effector caspases (12-16). Activation of caspase-3 requires proteolytic processing of its inactive zymogen/proform into activated p17 and p12 subunits (17). Procaspase-7 is activated through proteolytic processing by upstream caspases at Asp23, Asp198, and Asp206 to produce the mature subunits (14,16). Procaspase-6 is cleaved by caspase-3 at Asp23, Asp179 and Asp193 to form active large (p18) and small (p11) subunits (7).
PARP, a 116 kDa nuclear poly (ADP-ribose) polymerase, appears to be involved in DNA repair in response to environmental stress (18). This protein can be cleaved by many ICE-like caspases in vitro (2,19) and is one of the main cleavage targets of caspase-3 in vivo (17,20). In human PARP, the cleavage occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (17,19). PARP helps cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (21).
Lamins are nuclear membrane structural components that are important in maintaining normal cell functions such as cell cycle control, DNA replication, and chromatin organization (22-24). Lamin A/C is cleaved by caspase-6 and serves as a marker for caspase-6 activation. During apoptosis, lamin A/C is specifically cleaved into large (41-50 kDa) and small (28 kDa) fragments (24,25). The cleavage of lamins results in nuclear disregulation and cell death (26,27).
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