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Immunofluorescence analysis of Phospho-Inhibitor-2 pThr72 was done on 70% confluent log phase A431 cells treated with 3uM of Nocodazole for 24 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-Inhibitor-2 pThr72 Rabbit Polyclonal Antibody (441160G) at 2ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing punctuated nuclear localization. Panel e is untreated cell with no signal. Panel f is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human Inhibitor-2 that contains threonine 72. This sequence is conserved in rabbit, dog, zebrafish, chimpanzee, cow, and chicken.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2-3 µg/mL|
|Immunofluorescence (IF)||2-3 µg/mL|
|Immunohistochemistry (IHC)||Assay Dependent|
|Western Blot (WB)||1:1000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Inhibitor-2 (I-2) exists as a heterodimer with protein phosphatase type-1 (PP1), termed MgATP-dependent phosphatase. I-2 binds and forms an inactive complex with PP1 in its unphosphorylated state. This complex is activated through a series of phosphorylations on serine and threonine residues in I-2 that increases phosphatase activity. Multiple kinases have been implicated in phosphorylation of I-2 at threonine 72, namely GSK-3, cdc2 and ERK1. Casein kinase II phosphorylates I-2 at serine residues, which in turn enhances threonine phosphorylation by GSK-3. Recent evidence has shown that phosphorylation at threonine 72 peaks during prophase of the cell cycle and is localized in the centrosomes. The I-2/PP1 complex also binds neurabin and the kinases Nek2, KPI-2, and Aurora-A. Regulation of I-2/PP1 has been shown to be important in cell cycle, gene expression, ion gating, and neuromodulation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
IPP2; phosphoprotein phosphatase; PPP1R2; protein phosphatase 1, regulatory (inhibitor) subunit 2
IPP-2; IPP2; PPP1R2