Flow cytometric analysis of Jurkat cells, U0126 (10uM, 2hr; blue) or treated with TPA (200uM, 30min; green) using Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 647 Conjugate) (solid lines) or concentration-matched
Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (dashed lines).
|REACTIVITY||H M R Mk|
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb #12032.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised August 2019
Protocol Id: 407
Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of RSK1, RSK2, and RSK3 proteins only when phosphorylated at Ser380 (RSK1), Ser386 (RSK2), or Ser377 (RSK3).Species Reactivity:
Human, Mouse, Rat, MonkeySpecies predicted to react based on 100% sequence homology:
Chicken, Xenopus, Zebrafish, Bovine, Dog, Pig, Horse
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser377 of human p90RSK3 protein.
The 90 kDa ribosomal S6 kinases (RSK1-4) are a family of widely expressed Ser/Thr kinases characterized by two nonidentical, functional kinase domains (1) and a carboxy-terminal docking site for extracellular signal-regulated kinases (ERKs) (2). Several sites both within and outside of the RSK kinase domain, including Ser380, Thr359, Ser363, and Thr573, are important for kinase activation (3). RSK1-3 are activated via coordinated phosphorylation by MAPKs, autophosphorylation, and phosphoinositide-3-OH kinase (PI3K) in response to many growth factors, polypeptide hormones, and neurotransmitters (3).
Upon mitogenic stimulation, p44/42 ERK1/2 and ERK5 MAP kinases cooperatively phosphorylate p90RSK at Thr573 (p90RSK1 numbering) located within the carboxy-terminal kinase domain and at Thr359/Ser363 in the linker region between the two kinase domains (3). Phosphorylation of p90RSK at Thr573 within the activation loop of the p90RSK carboxy-terminal kinase domain promotes activation and directs phosphorylation of Ser380 within the hydrophobic stretch of the linker region (4,5). The p90RSK phosphorylated at Ser380 acts as a docking site for the constitutively active Ser/Thr kinase PDK1, which in turn phosphorylates Ser221 within the amino-terminal kinase domain activation loop, resulting in full enzymatic activation of the p90RSK (6). Antibodies against these phosphorylation sites are useful for understanding the kinetics and regulation of p90RSK activation.
For more information regarding the phospho-regulatory sites within each RSK isoform, including more information regarding the seminal studies demonstrating the complex phosphorylation cascades involved, please see the references herein and PhosphoSitePlus® (www.phosphosite.org).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.