Immunofluorescence analysis of NFkappaB / p65 was performed using 70% confluent log phase HeLa 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 NFkappaB / p65 Rabbit Polyclonal Antibody (Product # PA5-16758) at 2µg/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) 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 (Product # 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 cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Rabbit, Rat|
|Published species reactivity||Rat, Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||A synthetic peptide corresponding to internal domain of human NFkappaB p65|
|Storage buffer||PBS, pH 7.6, with 0.2% BSA|
|Contains||15mM sodium azide|
|Storage Conditions||4° C|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:20|
|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.
|Immunohistochemistry (IHC)||See 3 publications below|
PA5-16758 targets NFkappaB/p65 in IHC (P) and WB applications and shows reactivity with Human, Rabbit, and Rat samples.
The PA5-16758 immunogen is a synthetic peptide corresponding to internal domain of human NFkappaB p65.
NFkappaB is a heterodimer consisting of a 50kDa DNA binding subunit and a 65kDa transactivating subunit (RelA). The p50/p65 heterodimer is present in the cytoplasm in inactive form bound to its inhibitor, IkappaB. In unstimulated cells, nuclear localization signals present on p65 are masked by members of the IkappaB family of inhibitory proteins. Subsequent to cell stimulation, IkappaB undergoes phosphorylation, ubiquitination and degradation by a proteosome-dependent pathway, allowing nuclear translocation of the active dimeric NFkappaB transcription factor.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Pretreatment with mineralocorticoid receptor blocker reduces intestinal injury induced by ischemia and reperfusion: involvement of inhibition of inflammatory response, oxidative stress, nuclear factor ¿B, and inducible nitric oxide synthase.
PA5-16758 was used in immunohistochemistry to study the roles of oxidative stress, NFkB and iNOS in the mechanism by which pretreatment with a mineralocorticoid receptor blocker protects against ischemia-reperfusion-induced intestinal injury
|Ozacmak HS,Ozacmak VH,Barut F,Arasl¿ M,Ucan BH||The Journal of surgical research (191:350)||2014|
NF-¿B and COX-2 expression in nonmalignant endometrial lesions and cancer.
PA5-16758 was used in immunohistochemistry to perform a comparative study of the immunohistochemical expression of NFkB and COX-2 in non-malignant endometrial lesions and in endometrioid endometrial cancer
|Faloppa CC,Baiocchi G,Cunha IW,Fregnani JH,Osorio CA,Fukazawa EM,Kumagai LY,Badiglian-Filho L,Pinto GL,Soares FA||American journal of clinical pathology (141:196)||2014|
Mechanisms of the protective effects of curcumin against indomethacin-induced gastric ulcer in rats.
PA5-16758 was used in immunohistochemistry to study the molecular mechanisms underlying the protective effects of curcumin in a rat gastric ulcer model
|Morsy MA,El-Moselhy MA||Pharmacology (91:267)||2013|