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Technical Data Sheet


Sphingosine Kinase Activation
Antibody Sampler Kit


Kit Components
Applications: WB = Western blot, E = ELISA, ICC = Immunocytochemistry, IP = Immunoprecipitation, IHC = Immunohistochemistry Species: H = Human, R = Rat, M = Mouse, C = Chicken, F = Fish

Product References
SP4621 Neubauer, H & Pitson, SM (2016) F1000Research. 5:2825. IP-WB, ICC: HEK293, HeLa, MEF
SP4621 Wang, X. et al. (2016) PLoS Pathog. 12(10):e1005926. WB: Brain endothelial cells
SP4631 Wang, X. et al. (2016) PLoS Pathog. 12(10):e1005926. WB: Brain endothelial cells
SP4621 St Patrick, R. et al. (2015) Emerg micro & infect. 4(10):e61 ICC: hSKMC, MCF10A
SP1641 Durham, J. et al. (2015) Invest Ophthal Vis Sci. 56(6):3441 WB: human retinal cells and pericytes
SP1641 Sun, K. et al. (2015)Blood 125(10): 1643. WB: mouse erythrocyte
SP1641 Wilson, P.C. et al. (2015) Mol Endocrinol. 29(6):896 WB: rat aortic vascular smooth muscle cells
SP1641 Xiu, L. et al. (2015) Am J Pathol. 185(2):387 WB: hMSC cells
SP4621 Brunno, G et al. (2015) BBA MCB Lipids. 1851.2:194. WB: murine myoblast
SP4621 Wallington-Beddoe, CT et al. (2014) Cancer Res 74(10):2803. WB: ALL cell lines, K562 siRNA KD
SP4631 Wallington-Beddoe, CT et al. (2014) Cancer Res 74(10):2803. WB: ALL cell lines
SP1641 Kim, E.Y. et al. (2014) FASEB J. 28(10): 4347 WB: HEK293-CD40 cells
SP1641 Dai, L. et al. (2013) Molec Endocrinology 28(2):197. WB: human MCF7 breast cancer
SP4631 Dai, L. et al. (2013) Molec Endocrinology 28(2):197. WB: MCF7 + Insulin
SP1641 Park, K. et al. (2013) Mol Cell Biol. 33(4)752. WB: human keratinocytes
SP1641 Yasuo, M. et al. (2013) PLoS One 8(1):e53927. WB: rat lung
SP1621 Chakraborty, G. et al. (2012) J Neurochem. 121(5):806. WB: rat forebrain
SP1621 Yang, L. et al. (2012) Am J Pathol. 181(1):85. WB, ICC: mouse bone marrow stem cells
SP1641 Alvarez, S.E. et al. (2010) Nature 465:1084. WB: A7 melanoma cells
SP1641 Miller et al. (2008) Mol Cell Biol 28(12):4142. WB: human esophageal carcinoma
SP1641 Paugh, B.S. et al. (2008) FASEB J. 22(2):455. WB: human glioblastoma A172
SP1641 Shida, D. et al. (2008) Cancer Res. 68(16):6569. WB: human MNK1 cells + LPA
SP1621 Tao, R. et al. (2007) Cardiovas. Res. 74:56. WB: mouse heart, wildtype vs null
Kit Summary
The Sphingosine Kinase Activation kit can be used to detect activation of SK1 and SK2. The kit includes phospho-specific antibodies for detection of SK1 (Ser-225) and SK2 (Thr-578) phosphorylation, and anti-SK1 and anti-SK2 antibodies for comparison of total SK expression to the level of phosphorylation.

Western blot image of his-tagged human SK1 protein that was phosphorylated with ERK2. Blots were probed with anti-SK1 (lanes 1-3) and anti-SK1 (Ser-225) (lanes 4-6) in the absence (lanes 1 & 4) or presence of phospho-SK1 (Ser-225) (lanes 2 & 5), or unphosphorylated SK1 (Ser-225) (lanes 3 & 6) peptides.


Western blot of HeLa treated with calyculin A (lanes 1-4). The blots were untreated (lane 1 & 3) or treated with lambda phosphatase (lane 2 & 4), then probed with anti-SK2 (N-terminal region) (lanes 1 & 2) or anti-SK2 (Thr-578) (lanes 3 & 4).

Sphingolipids are metabolized into bioactive products that include ceramide, sphingosine, and sphingosine-1-phosphate (S1P). Sphingosine Kinase (SK) catalyzes the phosphorylation of the lipid sphingosine, creating S1P. S1P subsequently signals through cell surface G protein-coupled receptors, as well as intracellularly, to modulate cell proliferation, survival, motility and differentiation. Two isoforms of SK have been identified, SK1 and SK2. The mRNA for both of these isoforms is widely expressed with SK1 expression highest in brain, heart, kidney, thymus, spleen and lung, while SK2 is highest in kidney and liver. SKs can be activated through growth factor, G protein-coupled, and immunoglobulin receptor signalling. Regulation of SK1 and SK2 activity may occur through phosphorylation. SK1 is phosphorylated at Ser-225 by ERK leading to increased activity and translocation to the plasma membrane. SK2 is phosphorylated in response to EGF, PKC activators, and phorbol esters. ERK1 can phosphorylate both Ser-351 and Thr-578, and non-phosphorylatable mutants of these sites suppress ERK1-mediated chemotaxis.

Background References
Melendez, A.J. et al. (2000) Gene 251(1) :19.
Pitson, S.M. et al. (2003) EMBOJ. 22(20) :5491.
Spiegel, S. & Milstien, S. (2003) Nat. Rev. Mol. Cell Biol. 4:397.
Hait, N.C. et al. (2005) J Biol. Chem. 280:29462.
Pitson, S.M. et al. (2005) J Exp. Med. 201(1) :49.
Hait, N.C. et al. (2007) J Biol. Chem. 282(16):12058.
Buffer and Storage
Rabbit polyclonal antibodies are supplied in phosphate-buffered saline (PBS), 50% glycerol, 1 mg/ml BSA, and 0.05% sodium azide. Store at –20°C. Stable for 1 year.
imageRelated Products
Sphingosine Kinase 1 Phospho-Regulation Antibody Sampler Kit

Sphingosine Kinase 2 Phospho-Regulation Antibody Sampler Kit

Sphingosine Kinase Activation Antibody Sampler Kit

unphosphorylated SK1 (Ser-225) Blocking Peptide

phospho-SK1 (Ser-225) Blocking Peptide

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