Flow cytometric analysis of K-562 cells, λ phosphatase treated (blue) or untreated (green), using Phospho-SirT1 (Ser47) Antibody compared to a nonspecific negative control antibody (red).
Western blot analysis from wild-type mouse embryo fibroblasts (MEF) (lane 1) or SirT1 knockout mouse embryo fibroblasts (MEF) (lane 2) using SirT1 (D1D7) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the SirT1 knockout mouse embryo fibroblasts (MEF) confirms specificity of the antibody for SirT1. Wild-type and knockout MEFs were a gift from Wenyi Wei at the Harvard Medical School.
Confocal immunofluorescent analysis of HeLa (positive; left), WT MEF (positive; middle), and SirT1 KO MEF (right) cells using SirT1 (D1D7) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). WT and KO MEF were kindly provided by Wenyi Wei, Harvard Medical School.
Western blot analysis of extracts from various cell lines using SirT2 (D4O5O) Rabbit mAb.
Western blot analysis of mitochondrial extracts from SirT3 wild-type (WT) mouse liver (lane 1) and knockout (KO) mouse liver (lane 2), and of whole cell extracts from rat liver (lane 3) and human kidney (lane 4), using SirT3 (D22A3) Rabbit mAb (upper) and AIF (D39D2) XP® Rabbit mAb #5318 (lower). The absence of signal in the SirT3 knockout mouse liver confirms the specificity of the antibody for SirT3
Western blot analysis of extracts from 293 cells, expressing either nontargeting shRNA (293 shNT) or shRNA targeting SirT5 (293 shSirT5), and HCT 116, Raw 264.7, and H-4-II-E cells using SirT5 (D8C3) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). 293 shNT and 293 shSirT5 cells were kindly provided by David Lombard, University of Michigan.
Confocal immunofluorescent analysis of HCT 116 cells, expressing either non-targeting shRNA (shNT) (left) or shSirT6 (right), using SirT6 (D8D12) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Western blot analysis of extracts from various cell lines using SirT7 (D3K5A) Rabbit mAb.
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 HT-1080 cells, untreated (left) and phosphatase-treated (right), using Phospho-SirT1 (Ser47) Antibody (green). Actin filaments have been labeled using DY-554 phalloidin (red).
Western blot analysis of extracts from various cell lines using SirT1 (D1D7) Rabbit mAb.
Western blot analysis of extracts from SirT2 wild-type (WT) and knockout (KO) mouse brain using SirT2 (D4O5O) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). SirT2 WT and KO mouse brain extracts were kindly provided by Dr. Gizem Donmez, Tufts University School of Medicine.
Western blot analysis of extracts from control HeLa cells (lane 1) or SirT3 knockout HeLa cells (lane 2) using SirT3 (D22A3) Rabbit mAb #5490 (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). The absence of signal in the SirT3 knockout hela cells confirms the specificity of the antibody for SirT3.
Immunoprecipitation of SirT6 from HCT 116 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 3) or SirT6 (D8D12) Rabbit mAb (lane 2). Lane 1 is 10% input. Western blot analysis was performed using SirT6 (D8D12) Rabbit mAb.
Western blot analysis of 293 cell lysates treated or untreated with λ phosphatase, using Phospho-SirT1 (Ser47) Antibody #2314 (upper) or SirT1 Antibody #2310 (lower).
Western blot analysis of extracts from various cell lines using SirT6 (D8D12) Rabbit mAb.
Western blot analysis of extracts from HCT 116 cells, expressing either non-targeting shRNA (shNT) or shSirT6, using SirT6 (D8D12) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from SirT6 wild-type (WT) and knockout (KO) mouse embryonic fibroblasts (MEF) using SirT6 (D8D12) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). SirT6 WT and KO MEF were kindly provided by Dr. David Lombard, University of Michigan.
|Phospho-SirT1 (Ser47) Antibody 2314||20 µl||
|SirT1 (D1D7) Rabbit mAb 9475||20 µl||
||H Mk M R||120||Rabbit IgG|
|SirT2 (D4O5O) Rabbit mAb 12650||20 µl||
||H Mk M R||39, 43||Rabbit IgG|
|SirT3 (D22A3) Rabbit mAb 5490||20 µl||
||H M R||28||Rabbit IgG|
|SirT5 (D8C3) Rabbit mAb 8782||20 µl||
||H M R||30||Rabbit IgG|
|SirT6 (D8D12) Rabbit mAb 12486||20 µl||
||H Mk M R||42, 36||Rabbit IgG|
|SirT7 (D3K5A) Rabbit mAb 5360||20 µl||
||H Mk M R||45||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The Sirtuin Antibody Sampler Kit provides an economical means of evaluating total levels of sirtuin proteins. The kit includes enough antibody to perform at least two western blot experiments with each primary antibody.
Each antibody in the Sirtuin Antibody Sampler Kit recognizes endogenous levels of its specific target. Activation state antibodies detect their intended targets only when phosphorylated at the indicated site.
Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Phe297 of human SirT1 protein, Pro205 of human SirT2, or Val130 of mouse SirT3 protein, and with recombinant proteins specific to full-length human SirT5 protein, full-length mouse SirT6, or the amino terminus of human SirT7 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser47 of human SirT1. Polyclonal antibodies are purified by Protein A and peptide affinity chromatography.
The Silent Information Regulator (SIR2) family of genes is a highly conserved group of genes that encode nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylases, also known as Class III histone deacetylases. The best characterized of these genes is Saccharomyces cerevisiae Sir2, which is involved in silencing of mating type loci, telomere maintenance, DNA damage response and cell aging (1). SirT1, the mammalian ortholog of Sir2, is a nuclear protein implicated in the regulation of apoptosis, cellular senescence, endocrine signaling, glucose homeostasis, aging, and longevity. SirT2, one of several mammalian Sir2 homologs, deacetylates α-tubulin on Lys40 and histone H4 on Lys16, and is implicated in cytoskeletal regulation and progression through mitosis (2,3). SirT2 protein is mainly cytoplasmic and is associated with microtubules and the HDAC6 tubulin deacetylase (2).
SirT3 exists in human cells in two forms, including a full-length, nuclear (44 kDa) protein and a processed (28 kDa) protein found exclusively in the mitochondria (4-6). Full-length SirT3 protein is processed in the mitochondrial matrix by mitochondrial matrix processing peptidase (MMP) (5). Both full-length and processed SirT3 are active enzymes that deacetylate histone H3 at Lys9 and histone H4 at Lys16 in vitro (4). SirT3 also deacetylates Lys642 of acetyl-CoA synthetase 2 (AceCS2) and activates AceCS2 activity in the mitochondria (7).
SirT5 is localized to the mitochondria and has been implicated in the regulation of cell metabolism (8,9). Nuclear SirT6 is a chromatin-associated protein that promotes normal maintenance of genome integrity as mediated by the base excision repair (BER) pathway (10-12). Mammalian SirT7 is localized to the nucleolus and is prominently expressed in hematopoietic cells, especially myeloid progenitor cells (13). SirT7 is recruited to chromatin by sequence-specific DNA binding transcription factors such as Elk-4, where it facilitates transcriptional repression through deacetylation of histone H3 at Lys18 (14).
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