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12747
Smad2/3 Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Smad2/3 Antibody Sampler Kit #12747

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Smad2/3 Antibody Sampler Kit: 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).

Smad2/3 Antibody Sampler Kit: Image 2

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.

Smad2/3 Antibody Sampler Kit: Image 3

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.

Smad2/3 Antibody Sampler Kit: Image 4

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.

Smad2/3 Antibody Sampler Kit: Image 5

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.

Smad2/3 Antibody Sampler Kit: 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).

Smad2/3 Antibody Sampler Kit: Image 7

Western blot analysis of extracts from control HeLa cells (lane 1) or HeLa cells with an apparent in-frame truncation mutation in the gene encoding SMAD3 (lane 2) using Smad3 (C67H9) Rabbit mAb #9523 (upper) or β-actin (D6A8) Rabbit mAb #8457 (lower). The change in SMAD3 molecular weight in the mutated HeLa cells is consistent with an in-frame deletion.

Smad2/3 Antibody Sampler Kit: 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).

Smad2/3 Antibody Sampler Kit: Image 9

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.

Smad2/3 Antibody Sampler Kit: Image 10

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.

Smad2/3 Antibody Sampler Kit: Image 11

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.

Smad2/3 Antibody Sampler Kit: Image 12

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).

Smad2/3 Antibody Sampler Kit: Image 13

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 chromosome 6 (upper), including CDKN1A (lower), a known target gene of Smad3 (see additional figure containing ChIP-qPCR data).

Smad2/3 Antibody Sampler Kit: Image 14

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 chromosome 20 (upper), including ID1 (lower), a known target gene of Smad4 (see additional figure containing ChIP-qPCR data).

Smad2/3 Antibody Sampler Kit: Image 15

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).

Smad2/3 Antibody Sampler Kit: Image 16

Flow cytometric analysis of HeLa cells using Smad2/3 (D7G7) 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 control.

Smad2/3 Antibody Sampler Kit: Image 17

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.

Smad2/3 Antibody Sampler Kit: 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).

Smad2/3 Antibody Sampler Kit: Image 19

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).

Smad2/3 Antibody Sampler Kit: Image 20

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

Smad2/3 Antibody Sampler Kit: Image 21

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.

Smad2/3 Antibody Sampler Kit: Image 22

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

Smad2/3 Antibody Sampler Kit: Image 23

Immunoprecipitation of Smad2/3 protein from HCT116 extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Smad2/3 (D7G7) XP®. Western blot analysis was performed using Smad2/3 (D7G7) XP®. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody.

Smad2/3 Antibody Sampler Kit: Image 24

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).

Smad2/3 Antibody Sampler Kit: Image 25

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.

Smad2/3 Antibody Sampler Kit: Image 26

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

Smad2/3 Antibody Sampler Kit: Image 27

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

Smad2/3 Antibody Sampler Kit: Image 28

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).

To Purchase # 12747T
Product # Size Price
12747T
1 Kit  (6 x 20 µl) $ 445

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 

Product Description

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.

Specificity / Sensitivity

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.

Source / Purification

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.

Background

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.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.