Immunofluorescence analysis of I-kappa-B-alpha was done on 70% confluent log phase NIH/3T3 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 I-kappa-B-alpha(ZI002) Mouse Monoclonal Antibody (397700) at 2ug/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 (Product # 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 is a merged image showing Cytoplasmic and Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Human|
|Host / Isotype||Mouse / IgG1, kappa|
|Immunogen||Synthetic peptide derived from the C-terminal region of the human I-kappa-B-alpha protein|
|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|
|Western Blot (WB)||Assay Dependent|
* 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|
NFKB1 or NFKB2 is bound to REL, RELA, or RELB to form the NFKB complex. The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA or NFKBIB, MIM 604495), which inactivate NF-kappa-B by trapping it in the cytoplasm. Phosphorylation of serine residues on the I-kappa-B proteins by kinases (IKBKA, MIM 600664, or IKBKB, MIM 603258) marks them for destruction via the ubiquitination pathway, thereby allowing activation of the NF-kappa-B complex. Activated NFKB complex translocates into the nucleus and binds DNA at kappa-B-binding motifs such as 5-prime GGGRNNYYCC 3-prime or 5-prime HGGARNYYCC 3-prime (where H is A, C, or T; R is an A or G purine; and Y is a C or T pyrimidine).
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Human||1 µg/ml||KU812 cells provide a novel in vitro model of the human IL-33/ST2L axis: functional responses and identification of signaling pathways.||Tare N,Li H,Morschauser A,Cote-Sierra J,Ju G,Renzetti L,Lin TA||Experimental cell research (316:2527)||2010|