Confocal immunofluorescent analysis of HeLa cells using Cofilin (D3F9) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
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).
Flow cytometric analysis of Jurkat cells, using LIMK2 (8C11) Rabbit mAb Antibody (blue) compared to a nonspecific negative control antibody (red).
Western blot analysis of extracts from various cell types using TESK1 (D49D4) Rabbit mAb.
Immunoprecipitation of ROCK1 from C6 extracts. Lane 1 is 10% input, lane 2 is ROCK1 (C8F7) Rabbit mAb, and lane 3 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900. Western blot analysis was performed with ROCK1 (C8F7) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Western blot analysis of extracts from various cell types using Chronophin/PDXP (C85E3) Rabbit mAb.
Immunoprecipitation of SSH1 from Jurkat cell extracts. Lane is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is SSH1 (E1K3W) Rabbit mAb. Western blot analysis was performed using SSH1 (E1K3W) Rabbit mAb.
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.
Western blot analysis of extracts from various cell types using Cofilin (D3F9) XP® Rabbit mAb.
Western blot analysis of COS cells, untreated or λ phosphatase-treated, using Phospho-Cofilin (Ser3) (77G2) Rabbit mAb (upper) or Cofilin Antiobdy #3312 (lower).
Western blot analysis of extracts from Colo201 and Jurkat cells, using LIMK2 (8C11) Rabbit mAb.
Western blot analysis of extracts from various cell types using ROCK1 (C8F7) Rabbit mAb.
Western blot analysis of extracts from various cell lines using SSH1 (E1K3W) Rabbit mAb.
Western blot analysis of NIH/3T3 cells, λ phosphatase-treated or untreated, and various other cell lines, using Phospho-Cofilin (Ser3) (77G2) Rabbit mAb.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human SSH1 protein (hSSH1-Myc/DDK; +), using SSH1 (E1K3W) Rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).
|Cofilin (D3F9) XP® Rabbit mAb 5175||20 µl||
||Dg H Mk M R||19||Rabbit IgG|
|Phospho-Cofilin (Ser3) (77G2) Rabbit mAb 3313||20 µl||
||B H Mk M R||19||Rabbit IgG|
|LIMK2 (8C11) Rabbit mAb 3845||20 µl||
||H Mk||70||Rabbit IgG|
|TESK1 (D49D4) Rabbit mAb 4655||20 µl||
|ROCK1 (C8F7) Rabbit mAb 4035||20 µl||
||H Mk M R||160||Rabbit|
|Chronophin/PDXP (C85E3) Rabbit mAb 4686||20 µl||
||B Hm H Mk M R||31||Rabbit IgG|
|SSH1 (E1K3W) Rabbit mAb 13578||20 µl||
||H M R||140||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
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 two western blot experiments per antibody.
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. 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. SSH1 (E1K3W) Rabbit mAb recognizes endogenous levels of total SSH1 protein. Based on the absence of sequence homology, this antibody is not expected to recognize SSH2 or SSH3.
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; with a synthetic phosphopeptide corresponding to residues surrounding Ser3 of human cofilin protein; or with residues surrounding Pro1018 of human SSH1 protein.
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).
Slingshot homolog 1 (SSH1) can also dephosphorylate LIMK kinases, suppressing LIMK phosphorylation of cofilin (28). In addition, SSH1 modulates actin dynamics by stabilizing F-actin and promoting actin bundling independent of its cofilin phosphatase activity (29). SSH1 activity is regulated by phosphorylation and protein-protein interaction through various signaling pathways (1). Binding of SSH1 to F-actin stimulates its cofilin phosphatase activity (30).
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