Cell Signaling Technology

Product Pathways - Cytoskeletal Signaling

SPAK Antibody #2281

Applications Reactivity Sensitivity MW (kDa) Source
W IF-F H M R Mk Endogenous 65 Rabbit

Applications Key:  W=Western Blotting  IF-F=Immunofluorescence (Frozen)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

SPAK Antibody detects endogenous levels of total SPAK protein. This antibody does not cross-react with OSR1 or other members of the GCK family.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser436 of human SPAK. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293, C2C12 and NBT-II cells, using SPAK Antibody.

IF-F

IF-F

Confocal immunofluorescent analysis of choroid plexus epithelial cells showing apical membrane localization, using SPAK Antibody (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Background

SPAK (STE20/SPS1-related Pro/Ala-rich kinase) and OSR1 (oxidative stress responsive 1) are members of the GCK family serine/threonine kinases. Overexpression and in vitro studies demonstrate that SPAK is able to activate p38 MAP kinase indicating a possible role for SPAK in the stress response (1). Yeast two-hybrid screening revealed that SPAK and OSR1 bind to Na-K-2Cl cotransporters NKCC1 and NKCC2 and K-Cl cotransporter KCC3 (2). WNK1 and WNK4 phosphorylate SPAK at Thr243/247 and Ser380 (3-5). Similarly, WNK1 and WNK4 phosphorylate OSR1 at Thr185 and Ser315 (3, 4). Phosphorylation at these sites stimulates SPAK and OSR1 activity, leading to NKCC1 phosphorylation and enhanced NKCC1 activity (3-5). SPAK is also phosphorylated at Ser311 by PKCθ in response to T cell activation. Substitution of Ser311 with Ala or specific siRNA knock-down of SPAK dramatically reduces TCR/CD28-induced AP-1 activation, suggesting SPAK is involved in T cell signaling as well (6).

  1. Johnston, A.M. et al. (2000) Oncogene 19, 4290-4297.
  2. Piechotta, K. et al. (2002) J. Biol. Chem. 277, 50812-50819.
  3. Vitari, A.C. et al. (2005) Biochem. J. 391, 17-24.
  4. Moriguchi, T. et al. (2005) J. Biol. Chem. 280, 42685-42693.
  5. Gagnon, K.B. et al. (2006) Mol. Cell. Biol. 26, 689-698.
  6. Li, Y. et al. (2004) EMBO J. 23, 1112-1122.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

Products