Cell Signaling Technology

Product Pathways - Cytoskeletal Signaling

Actin Reorganization Antibody Sampler Kit #9967

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Cofilin (Ser3) (77G2) Rabbit mAb #3313 40 µl W IF-IC H M R Mk B 19 Rabbit IgG
Cofilin (D3F9) XP® Rabbit mAb #5175 40 µl W IF-IC H M R Mk Dg 19 Rabbit IgG
Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (41A3) Rabbit mAb #3149 40 µl W IHC-P IF-IC H M R Mk Dm B (X) (Dg) 75 Moesin. 80 Ezrin, Radixin. Rabbit IgG
Ezrin/Radixin/Moesin Antibody #3142 40 µl W IHC-P H M R Mk B (X) 75 Moesin. 80 Ezrin and Radixin. Rabbit
Phospho-VASP (Ser157) Antibody #3111 40 µl W H M R Mk GP 50 Rabbit
Phospho-VASP (Ser239) Antibody #3114 40 µl W H M R Mk 48, 50 Rabbit
VASP (9A2) Rabbit mAb #3132 40 µl W IP IF-IC H M R Hm Mk B 46, 50 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  Dm=D. melanogaster  X=Xenopus  B=Bovine  Dg=Dog  GP=Guinea Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

All activation state antibodies only detect their target proteins when modified at the indicated site. Cofilin (D3F9) XP® Rabbit mAb detects endogenous levels of total cofilin protein. VASP (9A2) Rabbit mAb detects endogenous levels of total VASP protein. Neither of the Ezrin/Radixin/Moesin antibodies cross-react with related phosphoproteins such as merlin or band 4.1.

Western Blotting

Western Blotting

Western blot analysis of extracts from A-431 cells, untreated (-) or treated with Forskolin #3828 (+), using Phospho-VASP (Ser157) Antibody #3111 (upper) and VASP (9A2) Rabbit mAb #3132 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from A-431 cells, untreated (-) or treated with Forskolin #3828 (+), using Phospho-VASP (Ser239) Antibody #3114 (upper) and VASP (9A2) Rabbit mAb #3132 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using VASP (9A2) Rabbit mAb.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell types using Ezrin/Radixin/Moesin Antibody #3142.

Western Blotting

Western Blotting

Western blot analysis of untreated or λ phosphatase treated A431 cells using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (41A3) Rabbit mAb #3149 (upper) and Ezrin/Radixin/Moesin Antibody #3142 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from untreated or λ-phosphatase-treated COS and HeLa cells using Phospho-Cofilin (Ser3) (77G2) Rabbit mAb #3313.


Western Blotting

Western Blotting

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

Description

The Actin Reorganization Antibody Sampler Kit contains reagents to examine proteins that help regulate the dynamic actin cytoskeleton. This kit includes enough primary and secondary antibodies to perform four Western blot experiments with each primary antibody.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to central residues of human cofilin1 and residues near the carboxy terminus of human and mouse VASP. Monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser3 of human cofilin and Thr567 of human ezrin. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to Thr567 of human ezrin. Activation state polyclonal antibodies are produced by immunizing rabbits with synthetic phosphopeptides corresponding to residues surrounding Ser157 and Ser239 of human VASP. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

Ubiquitous actin protein comprises the major structural component of the eukaryotic cytoskeleton. The formation and continual reorganization of the actin cytoskeleton is a key step in many biological processes, including cell motility, cytokinesis, endocytosis, embryonic development, tissue regeneration and the stress response (1). The small protein cofilin is one of a conserved family of actin-binding proteins that promote actin filament regeneration by severing preexisting filaments (2). Phosphorylation of cofilin at Ser3 by LIMK or TESK inhibits cofilin severing activity (3-5). Ezrin, radixin, and moesin (ERM) proteins function as linker proteins and signal transducers between the plasma membrane and actin cytoskeleton. These proteins are involved in cell adhesion, membrane ruffling, and microvilli formation (6,7). Interactive cytosolic ERM proteins exist as monomers or dimers that form both intra- and intermolecular associations through their amino- and carboxy-terminal domains (8). Phosphorylation at carboxy-terminal threonine residues (Thr567 of ezrin, radixin at Thr564 and Thr558 of moesin) may alter protein conformation and disrupt these protein associations and result in ERM protein activation (9,10). Vasodilator-stimulated phosphoprotein (VASP) is an adaptor protein that links the cytoskeleton with signal transduction pathways to act in fibroblast migration, platelet activation and axon guidance (11,12). Three phosphorylation sites (Ser157, Ser239, and Thr278) have been identified, with phosphorylation of Ser239 by PKG serving as a marker for nitric oxide and cGMP signaling (13). VASP Ser157 can act as a substrate for both PKA and PKC (14,15). Active VASP appears to promote actin polymerization by restricting actin filament capping, with PKA phosphorylation inhibiting this anti-capping activity (16).

  1. Carlier, M.F. et al. (1999) J. Biol. Chem. 274, 33827-33830.
  2. Condeelis, J. (2001) Trends Cell Biol. 11, 288-293.
  3. Arber, S. et al. (1998) Nature 393, 805-809.
  4. Yang, N. et al. (1998) Nature 393, 809-812.
  5. Toshima, J. et al. (2001) J. Biol. Chem. 276, 31449-31458.
  6. Louvet-Vallée, S. (2000) Biol. Cell 92, 305-316.
  7. Ivetic, A. and Ridley, A.J. (2004) Immunology 112, 165-176.
  8. Matsui, T. et al. (1998) J. Cell Biol. 140, 647-657.
  9. Gautreau, A. et al. (2000) J. Cell Biol. 150, 193-203.
  10. Tran Quang, C. et al. (2000) EMBO J. 19, 4565-4576.
  11. Ball, L.J. et al. (2000) EMBO J. 19, 4903-4914.
  12. Machesky, L.M. (2000) Cell 101, 685-688.
  13. Ibarra-Alvarado, C. et al. (2002) Mol. Pharmacol. 61, 312-319.
  14. Smolenski, A. et al. (1998) J. Biol. Chem. 273, 20029-20035.
  15. Chitaley, K. et al. (2004) FEBS Lett. 556, 211-215.
  16. Barzik, M. et al. (2005) J. Biol. Chem. 280, 28653-28662.

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