Western blot analysis of A431 cell extracts, untreated (-) or EGF treated (+), using Phospho-Rac1/cdc42 (Ser71) Antibody (upper) or Rac1/Cdc42 Antibody #4651 (lower).
Western blot analysis of extracts from various cell lines using Rac1/Cdc42 Antibody.
Confocal immunofluorescent analysis of HeLa cells, serum-starved (left) or EGF-treated #9908 (right), using Wave-2 (D2C8) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from various cell types using Profilin-1 (C56B8) Rabbit mAb.
Western blot analysis of extracts from various cell types using ARP2 Antibody.
Western blot analysis of extracts from HeLa, Jurkat and COS cells using ARP3 Antibody.
Immunoprecipitation of N-WASP from MCF-7 cells, using N-WASP (30D10) Rabbit mAb. Western blot was performed using the same antibody.
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.
Immunoprecipitation of WAVE-2 from HeLa cells using WAVE-2 (D2C8) XP® Rabbit mAb. Western blot was performed using the same antibody. Lane 1 is 5% input.
Western blot analysis of extracts from various cell types, using N-WASP (30D10) Rabbit mAb.
Western blot analysis of extracts from various cell types using WAVE-2 (D2C8) XP® Rabbit mAb.
|Phospho-Rac1/cdc42 (Ser71) Antibody 2461||20 µl||
|Rac1/Cdc42 Antibody 4651||20 µl||
||H Mk M R||21||Rabbit|
|WAVE-2 (D2C8) XP® Rabbit mAb 3659||20 µl||
||H Mk M R||80||Rabbit IgG|
|Profilin-1 (C56B8) Rabbit mAb 3246||20 µl||
||Dg Hm H Mk M R||15||Rabbit IgG|
|ARP2 Antibody 3128||20 µl||
||Dm Hm H Mk M R||44||Rabbit|
|ARP3 Antibody 4738||20 µl||
|N-WASP (30D10) Rabbit mAb 4848||20 µl||
||B Hm H Mk M R||65||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The Actin Nucleation and Polymerization Antibody Sampler Kit provides an economical means to evaluate the presence and status of actin nucleation and polymerization. The kit contains enough primary antibody to perform two western blots per primary.
ARP2 Antibody recognizes endogenous levels of total ARP2 protein. ARP3 Antibody recognizes endogenous levels of total ARP3 protein. This antibody does not cross-react with endogenous ARP2. Profilin-1 (C56B8) Rabbit mAb recognizes endogenous levels of total profilin-1 protein. This antibody may cross-react with profilin-2. Phospho-Rac1/cdc42 (Ser71) Antibody recognizes endogenous levels of Rac1/cdc42 only when phosphorylated at serine 71. This antibody may also detect phospho-RhoA (Ser73). Rac1/Cdc42 Antibody recognizes endogenous levels of total rac1 and cdc42 proteins. Based on sequence similarities, this antibody may also detect rac2 and rac3; this antibody does not cross-react with RhoA, RhoB, or RhoC. N-WASP (30D10) Rabbit mAb recognizes endogenous levels of total N-WASP protein. This antibody does not cross-react wth the hematopietic protein, WASP. WAVE-2 (D2C8) XP® Rabbit mAb recognizes endogenous levels of total WAVE-2 protein.
Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human profilin-1 protein, the sequence of human N-WASP protein, or central residues of human WAVE-2 protein.
Activation state specific polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser71 of human Rac1/cdc42 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino termini of human rac1 and cdc42 proteins, the sequence of human ARP2 protein, or the amino terminus of human ARP3 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Actin nucleation is the process of forming new actin filaments and is necessary to stimulate actin polymerization. Actin polymerization is vital for cell motility, cell division, and cell adhesion. Rac and Cdc42, members of the Rho-GTPase family, play key roles in actin dynamics, membrane trafficking, transcriptional regulation, cell growth, and development (1). GTP binding stimulates the activity of Rac/Cdc42, and the hydrolysis of GTP to GDP through the intrinsic GTPase activity or Rac/Cdc42, rendering it inactive. GTP hydrolysis is aided by GTPase activating proteins (GAPs), while exchange of GDP for GTP is facilitated by guanine nucleotide exchange factors (GEFs). Another level of regulation is achieved through binding of RhoGDI, a guanine dissociation inhibitor, which retains Rho family GTPases, including Rac and Cdc42, in their inactive GDP-bound state (2,3). Hematopoietic WASP and ubiquitously expressed N-WASP are autoinhibited in unstimulated cells. Upon stimulation they are activated by Cdc42, which relieves the autoinhibition in conjunction with phosphatidyl inositol 4,5-bisphosphate (4). Three WAVE (Wasf, SCAR) family proteins are similar in sequence to WASP and N-WASP, but lack the WASP/N-WASP autoinhibition domains and are indirectly activated by Rac (4). WAVE-2 is widely distributed, while WAVE-1 and WAVE-3 are strongly expressed in the brain (5). The highly conserved ARP2/3 complex is an important actin nucleation protein complex consisting of ARP2, ARP3, and ARPC1-5. The ARP2/3 complex promotes branching of existing actin filaments and formation of daughter filaments following activation by nucleation-promoting factors, such as WASP/WAVE or cortactin (6). Profilins are conserved actin binding proteins that affect the rate of actin polymerization by binding actin monomers and promoting exchange of ADP for ATP (reviewed in 7). Profilins bind to proteins involved in the regulation of actin dynamics including paladin (8), dynamin-I (9), VASP (10), and N-WASP (11).
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