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12747
Smad2/3 Antibody Sampler Kit
Primary Antibodies

Smad2/3 Antibody Sampler Kit #12747

Western Blotting Image 1

Western blot analysis of extracts from untreated or TGF-beta treated HeLa and NIH/3T3 cells, using Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb (upper), or Smad2 Antibody #3102 (lower).

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Western Blotting Image 2

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

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Chromatin IP-seq Image 3

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with hTGF-β3 #8425 (7 ng/ml, 1 hr) and Smad2/3 (D7G7) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ID1, a known target gene of Smad2/3 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 4

Western blot analysis of extracts from HeLa and ACHN cells using Smad2/3 (D7G7) XP® Rabbit mAb.

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Western Blotting Image 5

Western blot analysis of extracts from HT-1080, C2C12, or KNRK cells, untreated (-) or treated with TGF-β (10 ng/ml, 30 min; +), using Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb (upper) or total Smad3 (C67H9) Rabbit mAb #9523 (lower).

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Chromatin IP-seq Image 6

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with Human TGF-β3 #3706 (7 ng/ml) for 1 h and Smad3 (C67H9) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across CDKN1A, a known target gene of Smad3 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 7

Western blot analysis of extracts from HT1080 (human), C2C12 (mouse) and B35 (rat) using Smad3 (C67H9) Rabbit mAb.

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Chromatin IP-seq Image 8

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with TGF-β1 #8915 (7 ng/mL, 1 hr) and Smad4 (D3M6U) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ID1, a known target gene of Smad4 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 9

Western blot analysis of extracts from various cell lines using Smad4 (D3M6U) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). HT-29 and COLO 205 are Smad4-null mutant cell lines, confirming specificity of the antibody.

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Western Blotting Image 10

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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Flow Cytometry Image 11

Flow cytometric analysis of HeLa cells using Smad2 (D43B4) XP® Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

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Chromatin IP Image 12

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with hTGF-β3 #8425 (7 ng/ml, 1 hr) and either Smad2/3 (D7G7) XP® Rabbit mAb 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.

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Flow Cytometry Image 13

Flow cytometric analysis of HeLa cells using Smad2/3 (D7G7) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).

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Chromatin IP Image 14

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with Human TGF-β3 #3706 (7 ng/ml) for 1 h and either Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb 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, human c-Myc intron 1 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.

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Chromatin IP Image 15

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with Human TGF-β3 #3706 (7 ng/ml) for 1 h and either Smad3 (C67H9) Rabbit mAb 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.

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Western Blotting Image 16

Western blot analysis of extracts from HT1080 cells, treated with TGF-β1, TGFR inhibitor SB-431542 or BMP-2, using Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb #9520 (upper) or total Smad3 (C67H9) Rabbit mAb #9523 (lower).

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IP Image 17

Immunoprecipitation of Smad4 protein from HCT 116 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Smad4 (D3M6U) Rabbit mAb. Western blot analysis was performed using Smad4 (D3M6U) Rabbit mAb.

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Chromatin IP Image 18

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with TGF-β1 #8915 (7 ng/mL, 1 hr) and either Smad4 (D3M6U) Rabbit mAb 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 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 (equivalent to one).

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IF-IC Image 19

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).

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IF-IC Image 20

Confocal immunofluorescent analysis of HeLa cells, serum starved (left), treated with hTGF-β3 #8425 (100 ng/ml, 30 min, center), or treated with hTGF-β3 and SB43152 (10 ug/mL, 1 hr, right), using Smad2/3 (D7G7) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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Flow Cytometry Image 21

Flow cytometric analysis of HT-1080 cells using Smad3 (C67H9) Rabbit mAb #9523 (blue) compared to a nonspecific negative control antibody (red).

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Chromatin IP Image 22

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.

Learn more about how we get our images
IF-IC Image 23

Confocal immunofluorescent analysis of HT1080 cells, untreated (left) or TGFβ-treated (right), using Smad3 (C67H9) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb 3108 20 µl
  • WB
H M R Mi 60 Rabbit IgG
Smad2 (D43B4) XP® Rabbit mAb 5339 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R Mk 60 Rabbit IgG
Smad2/3 (D7G7) XP® Rabbit mAb 8685 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R Mk 52, 60 Rabbit IgG
Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb 9520 20 µl
  • WB
  • IP
  • ChIP
H M R 52 Rabbit IgG
Smad3 (C67H9) Rabbit mAb 9523 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R Mk 52 Rabbit IgG
Smad4 (D3M6U) Rabbit mAb 38454 20 µl
  • WB
  • IP
  • ChIP
H M R Mk 70 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Smad2/3 Antibody Sampler Kit provides an economical means of detecting target proteins of the TGF-β signaling pathway. The kit includes enough antibody to perform two western blots with each primary antibody.

Each antibody in the Smad2/3 Antibody Sampler Kit recognizes only its specific target and does not cross-react with other family members. Activation state antibodies detect their intended targets only when phosphorylated at the indicated site. The total Smad2, Smad3, and Smad4 antibodies detect their respective targets at endogenous levels.

Phospho-specific monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser465/467 of human Smad2 or Ser423/425 of human Smad3 protein. Total Smad2, Smad3, and Smad4 monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues near the amino termini of mouse Smad2 and Smad3, surrounding His198 of human Smad2/3, or surrounding Asp165 of human SMAD4 protein.

Transforming growth factor-β (TGF-β) superfamily signaling plays a critical role in the regulation of cell growth, differentiation, and development in a wide range of biological systems. In general, signaling is initiated with ligand-induced oligomerization of serine/ threonine receptor kinases and phosphorylation of the cytoplasmic signaling molecules Smad2 and Smad3 for the TGF-β/activin pathway, or Smad1/5/8 for the bone morphogenetic protein (BMP) pathway. Carboxy-terminal phosphorylation of Smad proteins by activated receptors results in their partnering with the common signaling transducer Smad4, and translocation to the nucleus. Activated Smad proteins regulate diverse biological effects by partnering with transcription factors resulting in cell-state specific modulation of transcription (1-4).

  1. Horbelt, D. et al. (2012) Int J Biochem Cell Biol 44, 469-74.
  2. Ikushima, H. and Miyazono, K. (2010) Nat Rev Cancer 10, 415-24.
  3. Kitisin, K. et al. (2007) Sci STKE 2007, cm1.
  4. Schmierer, B. and Hill, C.S. (2007) Nat Rev Mol Cell Biol 8, 970-82.
For Research Use Only. Not For Use In Diagnostic Procedures.

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