Immunofluorescence analysis of Phospho-SMAD2 pSer465 / pSer467 Antibody was done on 70% confluent log phase HeLa 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 treated with TGF-beta1(20ng/mL) for 15 minutes and labeled with Phospho-SMAD2 pSer465 / pSer467 Antibody(44244G) at 1:250 dilution in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa flour 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 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 594 Phalloidin (A12381). Panel d is a merged image showing Nuclear localization. Panel e showing merged image of untreated cells with less nuclear signals Panel f is a no primary antibody control. The images were captured at 40X magnification.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Human, Mouse, Not Applicable|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human Smad2 that contains serines 465 and 467. The sequence is conserved in 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 *|
|ChIP assay (ChIP)||10µl|
|Flow Cytometry (Flow)||1:20|
|Immunohistochemistry (Paraffin) (IHC (P))||1:20-1:200|
|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.
SMAD2, also known as MADH2 or MAD2 regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. Smad2 interacts with the TGF-beta receptors through its interaction with the SMAD anchor for receptor activation into the nucleus is a central event in TGF beta signaling. Phosphorylation of threonine 8 in the calmodulin-binding region of the MH1 domain by extracellular signalregulated kinase 1 (ERK1) enhances Smad2 transcriptional activity, which is negatively regulated by calmodulin.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||
Melanocytes Affect Nodal Expression and Signaling in Melanoma Cells: A Lesson from Pediatric Large Congenital Melanocytic Nevi.
44-244G was used in western blot to research pediatric large congenital melanocytic nevi to show melanocytes affect nodal expression and signaling in melanoma cells
|Margaryan NV,Gilgur A,Seftor EA,Purnell C,Arva NC,Gosain AK,Hendrix MJ,Strizzi L||International journal of molecular sciences (17:null)||2016|
BMP signaling controls muscle mass.
44-244G was used in western blot to demonstrate that bone morphogenetic protein signaling, acting through Smad, Smad5 and Smad8, is the fundamental hypertrophic signal in mice.
|Sartori R,Schirwis E,Blaauw B,Bortolanza S,Zhao J,Enzo E,Stantzou A,Mouisel E,Toniolo L,Ferry A,Stricker S,Goldberg AL,Dupont S,Piccolo S,Amthor H,Sandri M||Nature genetics (45:1309)||2013|
The significance of a Cripto-1 positive subpopulation of human melanoma cells exhibiting stem cell-like characteristics.
44-244G was used in western blot to investigate the role of cell surface CR- expression in human melanoma cells.
|Strizzi L,Margaryan NV,Gilgur A,Hardy KM,Normanno N,Salomon DS,Hendrix MJ||Cell cycle (Georgetown, Tex.) (12:1450)||2013|
Effects of a novel Nodal-targeting monoclonal antibody in melanoma.
44-244G was used in western blot to assess the use of 3D1 mAb for treating Nodal expressing cancers.
|Strizzi L,Sandomenico A,Margaryan NV,Focà A,Sanguigno L,Bodenstine TM,Chandler GS,Reed DW,Gilgur A,Seftor EA,Seftor RE,Khalkhali-Ellis Z,Leonardi A,Ruvo M,Hendrix MJ||Oncotarget (6:34071)||2015|