Western blot analysis was performed on whole cell extracts (30 ug lysate) of Jurkat (Lane 1), Serum Starved Jurkat (Lane 2), Jurkat Serum Starved for overnight following by serum release (Lane 3) and Jurkat treated for 15 min with 25 ng/ml of Calyculin A (lane 4). The blots were probed with Anti-RB1 [pS249]/[pT252] Rabbit Polyclonal Antibody (Product# 44584G, 1:500-1:2000 ug/ml) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # G21234, 1:5000 dilution). A 106 kDa band corresponding to RB1 [pS249]/[pT252] was observed across the treated cell lines tested. Two extra bands at 160 and 55 kDa were observed in treated cell lines. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Human|
|Published species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human Rb that contains serine 249 and threonine 252 (based on Swiss Protein database, accession number P06400). The sequence is conserved in human, mouse and rat.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/million cells|
|Western Blot (WB)||1:500-1:2000|
* 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 2 publications below|
Rb is a tumor suppressor nuclear phosphoprotein capable of binding to DNA. It is phosphorylated on serine and threonine, but not on tyrosine residues. It forms a complex with SV40 large T antigen, adenovirus E1A, and human papilloma virus-16 E. Rb protein may act by regulating transcription and loss of its function leads to uncontrolled cell growth. Aberrations in the RB gene have been implicated in cancers of breast, colon, prostate, kidney, nasopharynx, and leukemia. Phosphorylation of Rb at S608 depends on CDK4.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Human||Not Cited||Dual targeting of CDK and tropomyosin receptor kinase families by the oral inhibitor PHA-848125, an agent with broad-spectrum antitumor efficacy.||Albanese C,Alzani R,Amboldi N,Avanzi N,Ballinari D,Brasca MG,Festuccia C,Fiorentini F,Locatelli G,Pastori W,Patton V,Roletto F,Colotta F,Galvani A,Isacchi A,Moll J,Pesenti E,Mercurio C,Ciomei M||Molecular cancer therapeutics (9:2243)||2010|
|Mouse||Not Cited||Maid (GCIP) is involved in cell cycle control of hepatocytes.||Sonnenberg-Riethmacher E,Wüstefeld T,Miehe M,Trautwein C,Riethmacher D||Hepatology (Baltimore, Md.) (45:404)||2007|