Immunofluorescence analysis of CYLD was done on 70% confluent log phase SHSY5Y cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with CYLD Mouse Monoclonal Antibody (437700) at 1ug/mL in 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 localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Bovine, Horse, Human, Mouse, Non-human primate, Rat|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Recombinant protein derived from the N-terminus of human CYLD protein|
|Storage buffer||PBS, pH 7.4|
|Contains||0.1% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||1 µg/ml|
|Immunofluorescence (IF)||1 µg/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:10-1:50|
|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.
CYLD is a 956 aa, cytoplasmic, deubiquitinating enzyme belonging to the ubiquitin carboxy-terminal hydrolases (UCH) family of proteins with three cytoskeletal-associated protein-glycine-conserved (CAP-GLY) domains, a proline rich region, a SH3 binding domain and a sequence homology to catalytic domain of UCH. CYLD is identified as a tumor suppressor protein affecting the JNK signaling pathway. CYLD is a negative regulator of TRAF2 and NF-kappa-B signaling pathway and is also known to have receptor-dependent role in regulating the I-kappa-B kinase pathway. It also has a deubiquitinating activity that is directed towards non-Lys-48-linked polyubiquitin chains and TRAP1 is a novel substrate for deubiquitination. Mutated CYLD is known to be associated with cylindromatosis, multiple familial trichoepithelioma, and Brooke-Spiegler syndrome.
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