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12656
Smad 1/5/9 Antibody Sampler Kit

Smad 1/5/9 Antibody Sampler Kit #12656

Western Blotting Image 1

Western blot analysis of extracts from Hep G2 or MEF cells, untreated (-) or treated with Human BMP2 #4697 (50 ng/ml, 30 min; +), using Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad9 (Ser465/467) (D5B10) Rabbit mAb (upper) and Smad1 (D59D7) XP® Rabbit mAb #6944 (lower).

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Western Blotting Image 2

Western blot analysis of extracts from untreated or BMP-4-treated HeLa or NIH/3T3 cells using Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb.

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Western Blotting Image 3

Western blot analysis of extracts of HeLa cells, untreated or UV-treated (60 mJ/cm2 for 2 minutes followed by 1.5 hour recovery), using Phospho-Smad1 (Ser206) (D40B7) Rabbit mAb (upper) and Smad1 Antibody #9743 (lower).

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Western Blotting Image 4

Western blot analysis of extracts from various cell lines using Smad1 (D59D7) XP® Rabbit mAb.

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Chromatin IP-seq Image 5

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 6

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 7

Western blot analysis of extracts from various cell lines using Smad5 (D4G2) Rabbit mAb.

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Western Blotting Image 8

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 9

Flow cytometric analysis of HT-1080 cells, untreated (blue) or treated with Human BMP2 #4697 (green), using Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad9 (Ser465/467) (D5B10) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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Flow Cytometry Image 10

Flow cytometric analysis of HeLa cells, untreated (blue) or BMP-treated (green), using Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb compared to a nonspecific negative control antibody (red).

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Western Blotting Image 11

Western blot analysis of extracts from HT-1080 cells, untreated or treated with TPA #4174 (200 nM for 30 minutes), using Phospho-Smad1 (Ser206) (D40B7) Rabbit mAb (upper) and Smad1 Antibody #9743 (lower).

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Chromatin IP Image 12

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with Human BMP2 #4697 (50 ng/ml) for one hour and either Smad1 (D59D7) 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 ID1 Promoter Primers #5139, human SMAD6 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.

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IP Image 13

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 14

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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IP Image 15

Immunoprecipitation of Smad5 from HT-1080 cell extracts using Normal Rabbit IgG #2729 (lane 2) or Smad5 (D4G2) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Smad5 (D4G2) Rabbit mAb.

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IF-IC Image 16

Confocal immunofluorescent analysis of HT-1080 cells, serum-starved (overnight; left) or serum-starved and treated with Human BMP2 #4697 (50 ng/ml, 30 min; right), using Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad9 (Ser465/467) (D5B10) Rabbit mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 647 Conjugate) #8584 (red). Blue pseudocolor = Propidium Iodide (PI)/RNase Staining Solution #4087.

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IF-IC Image 17

Confocal immunofluorescent analysis of HT1080 cells, serum-starved (left) or serum-starved then treated with hBMP2 (50 ng/ml, 30 min; right) using Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb (green) and Cox IV (4D11-B3-E8) Mouse mAb #11967 (red).

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Chromatin IP Image 18

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with Human BMP2 #4697 (50 ng/ml, 1 hr) and either Smad5 (D4G2) 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 Smad6 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.

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IF-IC Image 19

Confocal immunofluorescent analysis of HT-1080 cells, BMP-treated (left) and untreated (right), using Phospho-Smad5 (Ser463/Ser465) (41D10) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad9 (Ser465/467) (D5B10) Rabbit mAb 13820 20 µl
  • WB
  • IP
  • IF
  • F
H M R 60 Rabbit IgG
Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb 9516 20 µl
  • WB
  • IF
  • F
H M R 60 Rabbit 
Phospho-Smad1 (Ser206) (D40B7) Rabbit mAb 5753 20 µl
  • WB
  • IP
H 60 Rabbit IgG
Smad1 (D59D7) XP® Rabbit mAb 6944 20 µl
  • WB
  • IP
  • ChIP
H M Mk 60 Rabbit IgG
Smad4 (D3M6U) Rabbit mAb 38454 20 µl
  • WB
  • IP
  • ChIP
H M R Mk 70 Rabbit IgG
Smad5 (D4G2) Rabbit mAb 12534 20 µl
  • WB
  • IP
  • ChIP
H M R Mk 60 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Smad1/5/9 Antibody Sampler Kit provides an economical means of detecting target proteins of the BMP signaling pathway. The kit includes enough antibody to perform two western blots with each primary antibody.

Each antibody in the Smad1/5/9 Antibody Sampler Kit recognizes endogenous levels of its respective target. Activation state antibodies detect their intended targets only when phosphorylated at the indicated site(s).

Phospho-specific monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser206 of human Smad1 protein or Ser463/465 of human Smad1 and Smad5 proteins. Total Smad1, Smad4, and Smad5 monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Ser190 of human Smad1, Asp165 of human SMAD4, or Pro249 of human Smad5 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 Smad1/5/9 for the bone morphogenetic protein (BMP) pathway. Carboxy-terminal phosphorylation of Smads by activated receptors results in their partnering with the common signaling transducer Smad4, and translocation to the nucleus. Activated Smads regulate diverse biological effects by partnering with transcription factors resulting in cell-state specific modulation of transcription (1-7) .

  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.
  5. Whitman, M. (1998) Genes Dev 12, 2445-62.
  6. Sapkota, G. et al. (2007) Mol Cell 25, 441-54.
  7. Alarcón, C. et al. (2009) Cell 139, 757-69.
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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Product # Size Price
12656T
1 Kit (6 x 20 µl) $ 423.0