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11958
PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 
Primary Antibodies
Antibody Duet

PhosphoPlus® Smad2 (Ser465/467) Antibody Duet  #11958

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other Image 1 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with Human TGF-β3 #8425 (7 ng/ml) for 1 h and either Smad2 (D43B4) XP® Rabbit mAb #5339 or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CDKN1A Intron 1 Primers #4669, SimpleChIP® Human ID1 Promoter Primers #5139, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

other Image 2 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Western blot analysis of extracts from HeLa cells (lane 1) or SMAD2 knock-out cells (lane 2) using Smad2 (D43B4) XP® Rabbit mAb #5339 (upper), and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). The absence of signal in the SMAD2 knock-out HeLa cells confirms specificity of the antibody for SMAD2.

other Image 3 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with Human Growth Factor β1 #8915 (7 ng/ml, 1 hr) and either Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human ID1 Promoter Primers #5139, Human JunB Promoter Primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

other Image 4 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Flow cytometric analysis of HeLa cells using Smad2 (D43B4) XP® Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

other Image 5 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Flow cytometric analysis of serum-starved HT-1080 cells, untreated (blue), treated with Human Transforming Growth Factor β3 (hTGF-β3) #8425 (100 ng/mL, 30 min; green) or pretreated with SB43152 (10 ug/mL, 30 min) and treated with hTGF-β3 (100 ng/mL, 30 min; red), using Phospho-Smad2 (Ser465/467) (E8F3R) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

other Image 6 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Confocal immunofluorescent analysis of NIH/3T3 cells, serum-starved (left) or treated with hTGF-β3 #8425 (right), using Smad2 (D43B4) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

other Image 7 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Confocal immunofluorescent analysis of serum-starved HT-1080 cells, untreated (left), treated with Human Transforming Growth Factor β3 (hTGF-β3) #8425 (100 ng/mL, 20 min; center), or treated with hTGF-β3 and post-processed with λ-phosphatase (right), using Phospho-Smad2 (Ser465/467) (E8F3R) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).

other Image 8 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Immunoprecipitation of phospho-SMAD2 (Ser465/Ser467) from extracts of HaCaT cells treated with Human Transforming Growth Factor β1 (hTGF-β1) #8915 (10nM, 30mins). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb. Western blot analysis was performed using P-SMAD2 (Ser465/ Ser467) (E8F3R) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used for detection to avoid cross-reactivity with IgG.

other Image 9 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Western blot analysis of extracts from various cell lines using Smad2 (D43B4) XP® Rabbit mAb.

other Image 10 - PhosphoPlus® Smad2 (Ser465/467) Antibody Duet 

Western blot analysis of extracts from HaCaT cells, untreated (-) or treated with Human Transforming Growth Factor β3 (hTGF-β3) #8425 (100ng/ml, 30mins) (+), using Phospho SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb (upper) and total Smad2 (D43B4) XP® Rabbit mAb, #5339 (lower).

To Purchase # 11958S
Product # Size Price
11958S
1 Kit $ 537

Product Includes Quantity Reactivity MW(kDa) Isotype
Smad2 (D43B4) XP® Rabbit mAb 5339 100 µl H M R Mk 60 Rabbit IgG
Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb 18338 100 µl H M R 60 Rabbit IgG

Product Description

PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.

Background

Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).

  1. Heldin, C.H. et al. (1997) Nature 390, 465-71.
  2. Attisano, L. and Wrana, J.L. (1998) Curr Opin Cell Biol 10, 188-94.
  3. Derynck, R. et al. (1998) Cell 95, 737-40.
  4. Massagué, J. (1998) Annu Rev Biochem 67, 753-91.
  5. Whitman, M. (1998) Genes Dev 12, 2445-62.
  6. Wu, G. et al. (2000) Science 287, 92-7.
  7. Attisano, L. and Wrana, J.L. (2002) Science 296, 1646-7.
  8. Moustakas, A. et al. (2001) J Cell Sci 114, 4359-69.
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus is a trademark of Cell Signaling Technology, Inc.