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9967
Actin Reorganization Antibody Sampler Kit

Actin Reorganization Antibody Sampler Kit #9967

Western Blotting Image 1

Western blot analysis of untreated or Staurosporine #9953 treated (1 uM, 3 hrs.) HeLa cells using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb #3726 (upper) and Ezrin/Radixin/Moesin Antibody #3142 (lower).

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Western Blotting Image 2

Western blot analysis of COS cells, untreated or λ phosphatase-treated, using Phospho-Cofilin (Ser3) (77G2) Rabbit mAb (upper) or Cofilin Antiobdy #3312 (lower).

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Western Blotting Image 3

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

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Western Blotting Image 4

Western blot analysis of various cell extracts, using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb.

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using Ezrin/Radixin/Moesin Antibody.

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Western Blotting Image 6

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

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Western Blotting Image 7

Western blot analysis of extracts from NIH/3T3 and C6 cells, untreated, 8-Br-cGMP-treated, 8-Br-cAMP-treated or forskolin-treated as indicated, using Phospho-VASP (Ser239) Antibody.

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Western Blotting Image 8

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

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Western Blotting Image 9

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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Western Blotting Image 10

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

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IF-IC Image 11

Confocal immunofluorescent analysis of HeLa cells using Cofilin (D3F9) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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Western Blotting Image 12

Western blot analysis of Staurosporine #9953 treated (1 μM, 3 hr) or untreated HeLa cells using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb (upper) and Ezrin/Radixin/Moesin Antibody #3142 (lower).

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Western Blotting Image 13

Western blot analysis of extracts from NIH/3T3 and C6 cells, untreated, 8-Br-cGMP-treated, 8-Br-cAMP-treated or forskolin-treated as indicated, using Phospho-VASP (Ser157) Antibody.

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Western Blotting Image 14

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

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IP Image 15

Immunoprecipitation of VASP from HeLa cell extracts using VASP Rabbit mAb. The same antibody was used for Western detection.

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IF-IC Image 16

Confocal immunofluorescent analysis of MCF7 cells either untreated (left) or λ phosphatase-treated (right), using Phospho-Cofilin (Ser3) (77G2) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 17

Immunohistochemical analysis of paraffin-embedded human breast ductal carcinoma using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb.

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IF-IC Image 18

Confocal immunofluorescent analysis of HUVE cells using VASP (9A2) Rabbit mAb (green) and MEK1/2 (L38C12) Mouse mAb #4694 (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

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IHC-P (paraffin) Image 19

Immunohistochemical analysis of paraffin-embedded human ovarian endometrioid adenocarcinoma using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb.

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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human gastric adenocarcinoma, untreated (left) or λ phosphatase treated (right), using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb.

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IF-IC Image 21

Confocal immunofluorescent analysis of HeLa cells, untreated (left), treated with Staurosporine #9953 (1 μM, 1hr; center), or λ phosphatase (right) labeled with Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 phalloidin (red). Blue pseudocolor = DRAQ5®#4084 (fluorescent DNA dye).

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ELISA-Peptide Image 22

Validation of Phospho-Ezrin (Thr567)/ Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb (ELISA Specific) in peptide DELFIA® assay using phospho- and nonphospho-peptide controls, and DELFIA® secondary antibodies (available from Perkin Elmer Life and Analytical Sciences).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Cofilin (Ser3) (77G2) Rabbit mAb 3313 20 µl
  • WB
  • IF
H M R Mk B 19 Rabbit IgG
Cofilin (D3F9) XP® Rabbit mAb 5175 20 µl
  • WB
  • IF
H M R Mk Dg 19 Rabbit IgG
Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb 3726 20 µl
  • WB
  • IHC
  • IF
H M R Mk 80 Rabbit IgG
Ezrin/Radixin/Moesin Antibody 3142 20 µl
  • WB
H M R Mk B 75 Moesin. 80 Ezrin and Radixin. Rabbit 
Phospho-VASP (Ser157) Antibody 3111 20 µl
  • WB
H M R Mk GP 50 Rabbit 
Phospho-VASP (Ser239) Antibody 3114 20 µl
  • WB
H M R Mk 48, 50 Rabbit 
VASP (9A2) Rabbit mAb 3132 20 µl
  • WB
  • IP
  • IF
H M R Hm Mk B 46, 50 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

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 two Western blot experiments with each primary antibody.

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.

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. 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.

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, 33827-30.
  2. Condeelis, J. (2001) Trends Cell Biol 11, 288-93.
  3. Arber, S. et al. (1998) Nature 393, 805-9.
  4. Matsui, T. et al. (1998) J Cell Biol 140, 647-57.
  5. Yang, N. et al. (1998) Nature 393, 809-12.
  6. Gautreau, A. et al. (2000) J Cell Biol 150, 193-203.
  7. Toshima, J. et al. (2001) J Biol Chem 276, 31449-58.
  8. Tran Quang, C. et al. (2000) EMBO J 19, 4565-76.
  9. Louvet-Vallée, S. (2000) Biol. Cell 92, 305-316.
  10. Ivetic, A. and Ridley, A.J. (2004) Immunology 112, 165-176.
  11. Ball, L.J. et al. (2000) EMBO J 19, 4903-14.
  12. Machesky, L.M. (2000) Cell 101, 685-8.
  13. Ibarra-Alvarado, C. et al. (2002) Mol. Pharmacol. 61, 312-319.
  14. Smolenski, A. et al. (1998) J Biol Chem 273, 20029-35.
  15. Chitaley, K. et al. (2004) FEBS Lett. 556, 211-215.
  16. Barzik, M. et al. (2005) J. Biol. Chem. 280, 28653-28662.
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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