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Immunofluorescence analysis of HAKAI was performed using 70% confluent log phase MCF-7 cells. 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 Hakai Rabbit Polyclonal Antibody (36-2800) 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 (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 represents the merged image showing nuclear and cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Dog, Human|
|Published species reactivity||Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide derived from the C-terminal region of the human Hakai protein.|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS, pH 7.4|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|ELISA (ELISA)||0.1-1 ug/mL|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Western Blot (WB)||2-4 µg/ml|
* 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.
|Western Blot (WB)||See 1 publications below|
Epithelial cell cadherin (CDH1; MIM 192090) is endocytosed as a consequence of tyrosine phosphorylation and ubiquitination. HAKAI is an E3 ubiquitin ligase (see UBE3A; MIM 601623) that mediates ubiquitination of the CDH1 complex.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Mouse||Not Cited||Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells.||Hanai J,Mammoto T,Seth P,Mori K,Karumanchi SA,Barasch J,Sukhatme VP||The Journal of biological chemistry (280:13641)||2005|
casitas B-lineage lymphoma-transforming sequence-like protein 1; HAKAI; RNF188