Immunofluorescence analysis of Phospho-p70S6K (Product # Thr229) was done on 70% confluent log phase A431 cells treated with 200ng of EGF for 10 minutes. 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 Phospho-p70S6K (Thr229) Rabbit Polyclonal Antibody (Product # 44-918G) at 1:250 dilution 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) 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 nuclear localization. Panel e is untreated cell with no signal. Panel f is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human p70-S6K that contains threonine 229. The sequence is conserved in rat.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:20|
|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.
p70 Ribosomal Protein S6 Kinase (p70S6K) is a 70 kDa member of the ribosomal S6 kinase (RSK) family of serine/threonine kinases. It is predominantly localized in the cytoplasm, and is essential in growth factors regulated cell proliferation, pathways involving cell motility, such as metastases, the immune response, and tissue repair. p70S6K acts downstream of phosphoinositide (PI) 3-kinase, and its main physiological target is the S6 ribosomal protein, which is involved in upregulation of protein synthesis.
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