Cell Signaling Technology

Product Pathways - Cytoskeletal Signaling

Cofilin Activation Antibody Sampler Kit #8354

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Cofilin (D3F9) XP® Rabbit mAb #5175 40 µl W IF-IC H M R Mk Dg 19 Rabbit IgG
Phospho-Cofilin (Ser3) (77G2) Rabbit mAb #3313 40 µl W IF-IC H M R Mk B 19 Rabbit IgG
Phospho-LIMK1 (Thr508)/LIMK2 (Thr505) Antibody #3841 40 µl W H (M) (R) 72 Rabbit
LIMK2 (8C11) Rabbit mAb #3845 40 µl W F H Mk (M) 70 Rabbit IgG
TESK1 (D49D4) Rabbit mAb #4655 40 µl W IP H 68 Rabbit IgG
ROCK1 (C8F7) Rabbit mAb #4035 40 µl W IP H M R Mk 160 Rabbit IgG
Chronophin/PDXP (C85E3) Rabbit mAb #4686 40 µl W H M R Hm Mk B 31 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  B=Bovine  Dg=Dog
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Cofilin (D3F9) XP® Rabbit mAb detects endogenous levels of total cofilin1 protein. Phospho-Cofilin (Ser3) (77G2) Rabbit mAb detects endogenous levels of cofilin only when phosphorylated at Ser3. Phospho-LIMK1 (Thr508)/LIMK2 (Thr505) Antibody detects transfected levels of LIMK1 and LIMK2 only when phosphorylated at Thr508 or 505. LIMK2 (8C11) Rabbit mAb detects endogenous levels of total LIMK2 protein and does not cross-react with LIMK1. TESK1 (D49D4) Rabbit mAb detects endogenous levels of total TESK1 protein. Chronophin/PDXP (C85E3) Rabbit mAb detects endogenous levels of total chronophin/PDXP protein. ROCK1 (C8F7) Rabbit mAb detects endogenous levels of total ROCK1 protein.

Western Blotting

Western Blotting

Western blot analysis of NIH/3T3 cells, λ phosphatase-treated or untreated, and various other cell lines, using Phospho-Cofilin (Ser3) (77G2) Rabbit mAb #3313.

Western Blotting

Western Blotting

Western blot analysis of extracts from COS-7 cells, untransfected (lane 1), transfected with Wild-type LIMK1 (lanes 2 and 3) or with LIMK1 T508A mutant (lanes 4 and 5), using Phospho-LIMK1 (Thr508)/LIMK2 (Thr505) Antibody #3841 (top), LIMK1 Antibody #3842 (middle) or HA-Tag (262K) mAb #2362 (bottom). Cells were either untreated (lanes 1, 2 and 4) or treated with PMA (lanes 3 and 5). (Triple HA-tagged LIMK1 plasmids kindly provided by Dr. K. Mizuno, Biological Institute, Tohoku University, Japan.)

Western Blotting

Western Blotting

Western blot analysis of extracts from Colo201 and Jurkat cells using LIMK2 (8C11) Rabbit mAb #3845.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using ROCK1 (C8F7) Rabbit mAb #4035.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using TESK1 (D49D4) Rabbit mAb #4655.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Chronophin/PDXP (C85E3) Rabbit mAb #4686.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Cofilin (D3F9) XP® Rabbit mAb #5175.

Description

The Cofilin Activation Antibody Sampler Kit provides an economical means to evaluate the presence and status of cofilin activation. The kit contains enough primary antibody to perform four western blot experiments per primary.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to central residues of human cofilin1 protein, carboxy-terminal residues of human LIMK2 protein, carboxy-terminal residues of human TESK1 protein, the central sequence of human ROCK1 protein; with recombinant mouse MBP-chronophin protein; or with a synthetic phosphopeptide corresponding to residues surrounding Ser3 of human cofilin protein. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr508 of human LIMK1 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

Cofilin and actin-depolymerization factor (ADF) are members of a family of essential conserved small actin-binding proteins that play pivotal roles in cytokinesis, endocytosis, embryonic development, stress response, and tissue regeneration (1). In response to stimuli, cofilin promotes the regeneration of actin filaments by severing preexisting filaments (2). The severing activity of cofilin is inhibited by LIMK or TESK phosphorylation at Ser3 of cofilin (3-5). Phosphorylation at Ser3 also regulates cofilin translocation from the nucleus to the cytoplasm (6).

LIM kinases (LIMK1 and LIMK2) are serine/threonine kinases that have two zinc finger motifs, known as LIM motifs, in their amino-terminal regulatory domains (7). LIM kinases are involved in actin cytoskeletal regulation downstream of Rho-family GTPases, PAKs, and ROCK (8,9). PAK1 and ROCK phosphorylate LIMK1 or LIMK2 at the conserved Thr508 or Thr505 residues in the activation loop, increasing LIMK activity (9-11). Activated LIM kinases inhibit the actin depolymerization activity of cofilin by phosphorylation at the amino-terminal Ser3 residue of cofilin (12,13).Testis-specific kinase 1 (TESK1) is an LIMK-related protein kinase originally identified to be highly expressed in testes and subsequently shown to be expressed in a wide variety of tissues and cell types (14-17). TESK1 phosphorylates the actin severing protein cofilin at Ser3, inactivating cofilin and thus regulating the organization of the actin cytoskeleton (15). Integrin signaling activates TESK1 activity and leads to stress fiber formation and cell spreading (15,18,19). TESK1 is involved in regulation of ERK signaling through its interaction with Spry2 (20) and regulation of cell spreading through its interaction with the focal adhesion protein actopaxin/α-parvin (18).Chronophin (CIN, PDXP) is a haloacid dehalogenase phosphatase that dephosphorylates cofilin. Alteration of CIN activity through overexpression of either the wildtype or phosphatase-inactive mutant CIN interferes with actin dynamics, cell morphology and cytokinesis (21).ROCK (Rho-associated kinase), a family of serine/threonine kinases, is an important downstream target of GTPase Rho and plays an important role in Rho-mediated signaling. Two isoforms of ROCK have been identified (ROCK1 and ROCK2). ROCK is composed of N-terminal catalytic, coiled-coil, and C-terminal PH (pleckstrin homology) domains. The C-terminus of ROCK negatively regulates its kinase activity (22,23). Caspase-3-induced cleavage of ROCK1 and direct cleavage of ROCK2 by granzyme B (grB) activates ROCK and leads to phosphorylation of myosin light chain and inhibition of myosin phosphatase (24). This phosphorylation may account for the mechanism by which Rho regulates cytokinesis, cell motility, cell membrane blebbing during apoptosis, and smooth muscle contraction (25-27).

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  2. Condeelis, J. (2001) Trends Cell Biol 11, 288-93.
  3. Arber, S. et al. (1998) Nature 393, 805-9.
  4. Yang, N. et al. (1998) Nature 393, 809-12.
  5. Toshima, J. et al. (2001) J Biol Chem 276, 31449-58.
  6. Nebl, G. et al. (1996) J Biol Chem 271, 26276-80.
  7. Okano, I. et al. (1995) J Biol Chem 270, 31321-30.
  8. Maekawa, M. et al. (1999) Science 285, 895-8.
  9. Edwards, D.C. et al. (1999) Nat Cell Biol 1, 253-9.
  10. Ohashi, K. et al. (2000) J Biol Chem 275, 3577-82.
  11. Sumi, T. et al. (2001) J Biol Chem 276, 670-6.
  12. Arber, S. et al. (1998) Nature 393, 805-9.
  13. Arber, S. et al. (1998) Nature 393, 805-9.
  14. Toshima, J. et al. (1995) J Biol Chem 270, 31331-7.
  15. Toshima, J. et al. (2001) Mol Biol Cell 12, 1131-45.
  16. Toshima, J. et al. (2001) J Biol Chem 276, 31449-58.
  17. Toshima, J. et al. (2001) Biochem Biophys Res Commun 286, 566-73.
  18. LaLonde, D.P. et al. (2005) J Biol Chem 280, 21680-8.
  19. Tsumura, Y. et al. (2005) Biochem J 387, 627-37.
  20. Chandramouli, S. et al. (2008) J Biol Chem 283, 1679-91.
  21. Gohla, A. et al. (2005) Nat Cell Biol 7, 21-9.
  22. Nakagawa, O. et al. (1996) FEBS Lett 392, 189-93.
  23. Lee, J.H. et al. (2004) J Cell Biol 167, 327-37.
  24. Sebbagh, M. et al. (2005) J Exp Med 201, 465-71.
  25. Amano, M. et al. (1996) J Biol Chem 271, 20246-9.
  26. Kureishi, Y. et al. (1997) J Biol Chem 272, 12257-60.
  27. Totsukawa, G. et al. (2000) J Cell Biol 150, 797-806.

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