Western blot analysis of HT-29 cells, untreated (-) or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP (Ser166) (D1L3S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Flow cytometric analysis of MCF7 cells using RIP (D94C12) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).
Western blot analysis of HT-29 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb (upper), or MLKL (D2I6N) Rabbit mAb #14993 (lower).
Western blot analysis of extracts from various cell lines using MLKL (D2I6N) Rabbit mAb. KARPAS cell Line source: Dr. Abraham Karpas at the University of Cambridge.
Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with etoposide #2200 (green), using Cleaved Caspase-3(Asp175) (5A1E) Rabbit mAb compared to a nonspecific negative control antibody (red).
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 SKW6.4 cells, untreated or anti-Fas-treated (1 µg/ml), using Cleaved Caspase-8 (Asp384) (11G10) Mouse mAb (left) or Caspase-8 (1C12) Mouse mAb #9746 (right).
Western blot analysis of extracts from HeLa, THP-1, and YB2/0 cell lines using Caspase-8 (D35G2) Rabbit mAb.
Western blot analysis of extracts from control HeLa cells (lane 1) or Caspase-8 knockout HeLa cells (lane 2) using Caspase-8 (D35G2) Rabbit mAb #4790 (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.
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.
Confocal immunofluorescent analysis of OVCAR8 cells using RIP (D94C12) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human MLKL protein (hMLKL-Myc/DDK; +), using MLKL (D2I6N) Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Confocal immunofluorescent images of HT-29 cells, untreated (left) or Staurosporine #9953 treated (right) labeled with Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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 CTLL-2 cells, untreated (-) or treated with Cycloheximide #2112 (CHX, 10 μg/ml, overnight) followed by Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/ml, 4 hr; +), using Caspase-8 (D35G2) Rabbit mAb.
Western blot analysis of extracts from various cell lines using RIP (D94C12) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse embryo, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb in the presence of control peptide (left) or Cleaved Caspase-3 (Asp175) Blocking Peptide (#1050) (right).
Western blot analysis of 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Caspase-8 protein (hCasp8-Myc/DDK; +), full-length mouse Caspase-8 protein (mCasp8; +), or full-length human Caspase-10 protein (hCasp10; +), using Caspase-8 (D35G2) Rabbit mAb.
Western blot analysis of extracts from HeLa cells, untransfected or transfected with human RIP construct, using RIP (D94C12) XP® Rabbit mAb.
Immunohistochemical analysis using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb on SignalSlide® Cleaved Caspase-3 IHC Controls #8104 (paraffin-embedded Jurkat cells, untreated (left) or etoposide-treated (right)).
Immunohistochemical staining of paraffin-embedded mouse embryo, showing cytoplasmic localization in apoptotic cells, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb.
Immunoprecipitation of extracts from Jurkat cells, untreated or etoposide-treated (25uM, 5hrs), using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb. Western blot was performed using the same antibody.
Western blot analysis of extracts from C6 (rat), NIH/3T3 (mouse), and Jurkat (human) cells, untreated or treated with staurosporine #9953 (1uM, 3hrs) or etoposide #2200 (25uM, 5hrs) as indicated, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb.
|Phospho-RIP (Ser166) (D1L3S) Rabbit mAb 65746||20 µl||
|RIP (D94C12) XP® Rabbit mAb 3493||20 µl||
||Hm H Mk M R||78||Rabbit IgG|
|Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb 91689||20 µl||
|MLKL (D2I6N) Rabbit mAb 14993||20 µl||
|Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb 9664||20 µl||
||H Mk M R||17, 19||Rabbit IgG|
|Caspase-3 (D3R6Y) Rabbit mAb 14220||20 µl||
||H Mk M R||35, 19, 17||Rabbit IgG|
|Cleaved Caspase-8 (Asp384) (11G10) Mouse mAb 9748||20 µl||
|Caspase-8 (D35G2) Rabbit mAb 4790||20 µl||
||H M R||10, 57||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
|Anti-mouse IgG, HRP-linked Antibody 7076||100 µl||
The Apoptosis/Necroptosis Antibody Sampler Kit provides an economical means of detecting markers for apoptosis and necroptosis. The kit contains enough primary antibody to perform at least two western blot experiments.
Each antibody in the Apoptosis/Necroptosis Antibody Sampler Kit detects endogenous levels of its target protein. MLKL (D2I6N) Rabbit mAb cross-reacts with a band at 130 kDa in some cell lines. Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb may also react with MLKL when dually phoshorylated at Thr357 and Ser358. Caspase-3 (D3R6Y) Rabbit mAb detects full-length caspase-3 as well as the large subunit (p20) of caspase-3 resulting from cleavage during apoptosis. Cleaved Caspase-3 (Asp175) (5A1) Rabbit mAb detects endogenous levels of the large subunit of caspase-3 but does not detect full-length caspase-3 or other cleaved caspases. Cleaved Caspase-8 (Asp384) (11G10) Mouse mAb detects endogenous levels of the small fragment of caspase-8 resulting from cleavage at aspartic acid 384. The antibody does not cross-react with full length caspase-8. Caspase-8 (D35G2) Rabbit mAb detects endogenous levels of total caspase-8, including the p10 subunit of the activated protein. It may also cross-react with overexpressed levels of caspase-10.
Monoclonal antibodies are produced by immunizing animals with synthetic peptides surrounding Leu190 of human RIP1, the carboxy terminus of human MLKL and human caspase-8, the amino-terminal sequence of p10 of human caspase-8, amino terminal residues adjacent to Asp175 of human caspase-3, a recombinant protein corresponding to the p20 subunit of caspase-3, and phosphopeptides surrounding human Ser166 of human RIP and Ser358 of human MLKL.
Apoptosis is a regulated physiological process leading to cell death (1,2). Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Caspases are synthesized as inactive zymogens containing a pro-domain followed by large (p20) and small subunits (p10) that are proteolytically processed in a cascade of caspase activity. Initiator caspases (including 8, 9, 10, and 12) are closely coupled to proapoptotic signals. 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. Cytochrome c released from mitochondria is coupled to the activation of caspase-9, a key initiator caspase. Apoptosis induced through the extrinsic mechanisms involving death receptors in the tumor necrosis factor receptor superfamily activates caspase-8. Activated caspase-8 cleaves and activates downstream effector caspases, such as caspase-1, -3, -6, and -7. Caspase-3 is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP).
Necroptosis, a regulated pathway for necrotic cell death, is triggered by a number of inflammatory signals, including cytokines in the tumor necrosis factor (TNF) family, pathogen sensors such as toll-like receptors (TLRs), and ischemic injury (3,4). Necroptosis is negatively regulated by caspase-8 mediated apoptosis in which the kinase RIP/RIPK1 is cleaved (5). Furthermore, necroptosis is inhibited by a small molecule inhibitor of RIP, necrostatin-1 (Nec-1) (6). Research studies show that necroptosis contributes to a number of pathological conditions, and Nec-1 has been shown to provide neuroprotection in models such as ischemic brain injury (7). RIP is phosphorylated at several sites within the kinase domain that are sensitive to Nec-1, including Ser14, Ser15, Ser161, and Ser166 (8). Phosphorylation drives association with RIP3, which is required for necroptosis (9-11). Mixed lineage kinase domain-like protein (MLKL) is a pseudokinase that was identified as downstream target of RIP3 in the necroptosis pathway (12). During necroptosis RIP3 is phosphorylated at Ser227, which recruits MLKL and leads to its phosphorylation at Thr357 and Ser358 (12). Knockdown of MLKL through multiple mechanisms results in inhibition of necroptosis (13). While the precise mechanism for MLKL-induced necroptosis is unclear, some studies have shown that necroptosis leads to oligomerization of MLKL and translocation to the plasma membrane, where it effects membrane integrity (14-17).
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