Western blot analysis of extracts from COS cells cotransfected with activated cdc42 and GST-PAK2 (lane 1), transfected with GST-PAK2 alone (lane 2) or activated cdc42 alone (lane 3), using Phospho-PAK1 (Ser144)/PAK2 (Ser141) Antibody (upper) or PAK1/2/3 Antibody #2604 (lower). This antibody detects GST-PAK2 signal only when it is cotransfected with activated cdc42, indicating that this antibody is phospho-specific.
Western blot analysis of extracts from guinea pig neutrophils stimulated with fMLP (1 µM ) for the indicated times, using Phospho-PAK1 (Ser199/204)/PAK2 (Ser192/197) Antibody.
Western blot analysis of extracts from guinea pig neutrophils stimulated with 1 µM fMLP for indicated times, using Phospho-PAK1 (Thr423)/PAK2 (Thr402) Antibody (Provided by Drs. Qian Zhan and John Badwey, Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Massachusetts.)
Western blot analysis of extracts from guinea pig neutrophils stimulated with fMLP for the indicated times, using Phospho-PAK2 (Ser20) Antibody (upper) or PAK1/2/3 Antibody #2604 (lower).
Immunoprecipitation of PAK1 from C6 and SH-SY5Y cells followed by Western blot analysis, using PAK1 Antibody.
Western blot analysis of extracts from 293, C2C12 and COS cells using PAK2 (C17A10) Rabbit mAb.
Immunoprecipitation of PAK3 from mouse brain extract, using PAK3 Antibody, followed by Western blot analysis using PAK3 Antibody (left), PAK1 Antibody #2602 (middle) or PAK2 Antibody #2608 (right).
Western blot analysis of extracts from SH-SY5Y, C6 and NIH/3T3 cells, using PAK1/2/3 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.
Western blot analysis of extracts from guinea pig neutrophils stimulated with fMLP for indicated times, using Phospho-PAK1 (Ser144)/PAK2 (Ser141) Antibody (upper) and PAK1/2/3 Antibody #2604 (lower).
Western blot analysis of extracts from HeLa cells treated with 0.4 M sorbitol for indicted times, using Phospho-PAK1 (Thr423)/PAK2 (Thr402) Antibody (upper) or PAK2 antibody (lower).
Western blot analysis of extracts from SH-SY5Y, C6 and NIH/3T3 cells, using PAK1 Antibody.
Western blot analysis of mouse brain extract and recombinant GST-PAK3, using PAK3 Antibody.
Western blot analysis of extracts from HeLa cells transfected with 100 nM control siRNA #6201 (-) or PAK1 siRNA, using PAK1 Antibody #2602 and p42 MAP Kinase (Erk2) Antibody #9108. The PAK1 Antibody confirms silencing of PAK1 expression, and p42 MAP Kinase (Erk2) Antibody is used to control for loading and specificity of PAK1 siRNA.
|Phospho-PAK1 (Ser144)/PAK2 (Ser141) Antibody 2606||20 µl||
||H M GP||61 to 67 (PAK2), 68 to 74 (PAK1/3)||Rabbit|
|Phospho-PAK1 (Ser199/204)/PAK2 (Ser192/197) Antibody 2605||20 µl||
||H M R GP||61 to 67 (PAK2), 68 to 74 (PAK1/3)||Rabbit|
|Phospho-PAK1 (Thr423)/PAK2 (Thr402) Antibody 2601||20 µl||
||H M GP||61 to 67 (PAK2), 68 to 74 (PAK1/3)||Rabbit|
|Phospho-PAK2 (Ser20) Antibody 2607||20 µl||
||H M GP||61 to 67||Rabbit|
|PAK1 Antibody 2602||20 µl||
||H M R Mk GP||68||Rabbit|
|PAK2 (C17A10) Rabbit mAb 2615||20 µl||
||H M Mk||61||Rabbit IgG|
|PAK3 Antibody 2609||20 µl||
||H M R||65||Rabbit|
|PAK1/2/3 Antibody 2604||20 µl||
||H M R Mk GP||61 (PAK2), 68 (PAK1/3)||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The PAK antibody sampler kit provides and economical means to evaluate the activation status of PAK1, 2, and 3. This kit includes enough primary and secondary antibodies to perform two western blots with each antibody.
Phospho-PAK2 (Ser20) Antibody, PAK1 Antibody, PAK2 (C17A10) Rabbit mAb, PAK3 Antibody and PAK1/2/3 Antibody detect endogenous levels of their target protein(s) and do not cross-react with other PAK family members. Phospho-PAK1 (Ser199/204)/PAK2 (Ser192/197), Phospho-PAK1 (Ser144)/PAK2 (Ser142), and Phospho-PAK1 (Thr423)/PAK2 (Thr402) antibodies, which may also detect Ser200/205, Ser139, and Thr421 of phosphorylated PAK3, respectively. Phospho-PAK1 (Thr423)/PAK2 (Thr402) Antibody cross-reacts with phospho-Mst1 (Thr183) or phospho-Mst2 (Thr180).
Phospho-specific antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser144, Ser199/204 and Thr423 of human PAK1, and Ser20 of human PAK2. PAK1 Antibody and PAK2 Antibody are produced by immunizing animals with synthetic peptides corresponding to residues surrounding the aminotermini of PAK1 and PAK2. PAK3 Antibody and PAK1/2/3 Antibody is produced by immunizing animals with a synthetic peptide corresponding to residues at the carboxy-terminus of human PAK3 and PAK1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
The p21-activated kinase (PAK) family of serine/threonine kinases is engaged in multiple cellular processes, including cytoskeletal reorganization, MAPK signaling, apoptotic signaling, control of phagocyte NADPH oxidase and growth factor-induced neurite outgrowth (1,2). Several mechanisms that induce PAK activity have been reported. Binding of Rac/cdc42 to the CRIB (or PBD) domain near the amino terminus of PAK causes autophosphorylation and conformational changes in PAK (1). Phosphorylation of PAK1 at Thr423 by PDK induces activation of PAK1 (3). Several autophosphorylation sites have been identified, including serines 199 and 204 of PAK1 and serines 192 and 197 of PAK2 (4,5). Because the autophosphorylation sites are located in the amino-terminal inhibitory domain, it has been hypothesized that modification in this region prevents the kinase from reverting to an inactive conformation (6). Research indicates that phosphorylation of Ser144 of PAK1 or Ser139 of PAK3 (located in the kinase inhibitory domain) affects kinase activity (7). Phosphorylation of Ser21 of PAK1 or Ser20 of PAK2 regulates binding with the adaptor protein Nck (8).
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