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.
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