The activation of caspases is a key regulatory step in apoptosis. Once cytochrome c is released from the mitochondria into the cytosol, it binds Apaf-1 to form an oligomeric cytochrome c/Apaf-1 complex, which induces caspase activation. Inhibitors of Apoptosis Proteins (IAPs), are a family of proteins that regulate the cytochrome c/Apaf-1 caspase activating pathway. Like cytochrome c, Smac (for second mitochondria-derived activator of caspase, also designated DIABLO in mouse for direct IAP binding protein with low PI) promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway by binding IAPs and preventing them from inhibiting caspases. In healthy cells, Smac is a mitochondrial protein, but when cells undergo apoptosis, Smac is released into the cytosol.
Background References
1. Masoumi KC et al. Identification of a novel protein kinase Cd-Smac complex that dissociates during paclitaxel-induced cell death. FEBS Lett 586:1166-72 (2012).
2. Lamers F et al. Identification of BIRC6 as a novel intervention target for neuroblastoma therapy. BMC Cancer 12:285 (2012).
Tissue Specificity
Ubiquitously expressed with highest expression in testis. Expression is also high in heart, liver, kidney, spleen, prostate and ovary. Low in brain, lung, thymus and peripheral blood leukocytes. Isoform 3 is ubiquitously expressed.
Western blot analysis of DIABLO on different lysates with Rabbit anti-DIABLO antibody (ET1610-74) at 1/2,000 dilution.
Lane 1: HeLa cell lysate (20 µg/Lane) Lane 2: Jurkat cell lysate (20 µg/Lane) Lane 3: C6 cell lysate (20 µg/Lane) Lane 4: Mouse testis tissue lysate (40 µg/Lane) Lane 5: Rat testis tissue lysate (40 µg/Lane)
Predicted band size: 27 kDa Observed band size: 21 kDa
Exposure time: 2 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1610-74) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunocytochemistry analysis of HeLa cells labeling DIABLO with Rabbit anti-DIABLO antibody (ET1610-74) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-DIABLO antibody (ET1610-74) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Counterstained with Mitotracker. Nuclear DNA was labelled in blue with DAPI.
Immunocytochemistry analysis of Neuro-2a cells labeling DIABLO with Rabbit anti-DIABLO antibody (ET1610-74) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-DIABLO antibody (ET1610-74) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Counterstained with Mitotracker. Nuclear DNA was labelled in blue with DAPI.
Immunocytochemistry analysis of C6 cells labeling DIABLO with Rabbit anti-DIABLO antibody (ET1610-74) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-DIABLO antibody (ET1610-74) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Counterstained with Mitotracker. Nuclear DNA was labelled in blue with DAPI.
Immunohistochemical analysis of paraffin-embedded human spleen tissue using anti-DIABLO antibody. Counter stained with hematoxylin.
Immunohistochemical analysis of paraffin-embedded mouse overy tissue using anti-DIABLO antibody. Counter stained with hematoxylin.
Immunohistochemical analysis of paraffin-embedded mouse skin tissue using anti-DIABLO antibody. Counter stained with hematoxylin.
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