Immunofluorescence analysis of NIX was performed using 70% confluent log phase K-562 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 BNIP3L (ZN002) Mouse Monoclonal Antibody (393300) at 2µg/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A28175) 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 membranous localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse, Non-human primate|
|Host / Isotype||Mouse / IgG1, kappa|
|Immunogen||Synthetic peptide derived from the internal region of human, mouse and rat Nix proteins|
|Storage buffer||PBS, pH 7.4|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay Dependent|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Western Blot (WB)||2 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.
Members in the Bcl-2 family are critical regulators of apoptosis by either inhibiting or promoting cell death. Bcl-2 homology 3 (BH3) domain is a potent death domain. BH3 domain containing pro apoptotic proteins, including Bad, Bax, Bid, Bik, Hrk, Nip3, and Bim, form a growing subclass of the Bcl-2 family. A novel BH3 domain containing protein was recently identified and designated Bnip3L, Bnip3a, and Nix (for Nip3-like protein X Bnip3L/Bnip3a/Nix is a homolog of the E1B 19K/Bcl-2 binding and pro-apoptotic protein Bnip3. Overexpression of Bnip3L induces apoptosis. Bnip3L interacts with and overcomes suppresses by Bcl-2 and Bcl-xL. Bnip3L is localized in mitochondria.
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