Revision 2

#12656Store at -20C

1 Kit

(6 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-SMAD1 (Ser463/465)/ SMAD5 (Ser463/465)/ SMAD9 (Ser465/467) (D5B10) Rabbit mAb 13820 20 µl 60 kDa Rabbit IgG
Phospho-SMAD1/5 (Ser463/465) (41D10) Rabbit mAb 9516 20 µl 60 kDa Rabbit 
Phospho-SMAD1 (Ser206) (D40B7) Rabbit mAb 5753 20 µl 60 kDa Rabbit IgG
SMAD1 (D59D7) XP® Rabbit mAb 6944 20 µl 60 kDa Rabbit IgG
SMAD4 (D3M6U) Rabbit mAb 38454 20 µl 70 kDa Rabbit IgG
SMAD5 (D4G2) Rabbit mAb 12534 20 µl 60 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

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 Smad1/5/9 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.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    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 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 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.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 2
    #12656

    SMAD 1/5/9 Antibody Sampler Kit

    SMAD 1/5/9 Antibody Sampler Kit: Image 1 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with BMP (50 ng/mL for 1 hour) and either Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad9 (Ser465/467) (D5B10) 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 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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from serum-starved 3T3 cells treated with hBMP2 (50ng/mL, 30 min) using Phospho-SMAD1/5 (Ser463/465) (41D10) Rabbit mAb. The virtual lane view (left) shows the target band (as indicated) and a band corresponding to Phospho-SMAD1/5 (Ser463/465) (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    SMAD 1/5/9 Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from various cell lines using SMAD5 (D4G2) Rabbit mAb.
    SMAD 1/5/9 Antibody Sampler Kit: Image 4 Expand Image
    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).
    SMAD 1/5/9 Antibody Sampler Kit: Image 5 Expand Image
    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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 6 Expand Image
    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).
    SMAD 1/5/9 Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using SMAD1 (D59D7) XP® Rabbit mAb.
    SMAD 1/5/9 Antibody Sampler Kit: Image 8 Expand Image
    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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 9 Expand Image
    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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 10 Expand Image
    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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 11 Expand Image
    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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 12 Expand Image
    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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 13 Expand Image
    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).
    SMAD 1/5/9 Antibody Sampler Kit: Image 14 Expand Image
    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).
    SMAD 1/5/9 Antibody Sampler Kit: Image 15 Expand Image
    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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 16 Expand Image
    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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 17 Expand Image
    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).
    SMAD 1/5/9 Antibody Sampler Kit: Image 18 Expand Image
    Confocal immunofluorescent analysis of HT-1080 cells, BMP-treated (left) and untreated (right), using Phospho-SMAD1/5 (Ser463/Ser465) (41D10) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
    SMAD 1/5/9 Antibody Sampler Kit: Image 19 Expand Image
    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).
    SMAD 1/5/9 Antibody Sampler Kit: Image 20 Expand Image
    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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 21 Expand Image
    Flow cytometric analysis of HT-1080 cells, untreated (blue) or treated with Human BMP-2 #4697 (50 ng/ml, 30 min; green) using Phospho-SMAD1/5 (Ser463/465) (41D10) 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.
    SMAD 1/5/9 Antibody Sampler Kit: Image 22 Expand Image
    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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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.