Revision 1

#9963Store at -20C

 

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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-SMAD1/5 (Ser463/465) (41D10) Rabbit mAb 9516 40 µl 60 kDa Rabbit 
SMAD1 (D59D7) XP® Rabbit mAb 6944 40 µl 60 kDa Rabbit IgG
SMAD5 Antibody 9517 40 µl 60 kDa Rabbit 
Phospho-SMAD2 (Ser465/467) (138D4) Rabbit mAb 3108 40 µl 60 kDa Rabbit IgG
Smad2 (D43B4) XP® Rabbit mAb 5339 40 µl 60 kDa Rabbit IgG
Phospho-SMAD3 (Ser423/425) (C25A9) Rabbit mAb 9520 40 µl 52 kDa Rabbit IgG
SMAD3 (C67H9) Rabbit mAb 9523 40 µl 52 kDa Rabbit IgG
Smad4 Antibody 9515 40 µl 70 kDa Rabbit 
Smad6 Antibody 9519 40 µl 62 kDa Rabbit 
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 Phospho-Smad Antibody Sampler Kit contains reagents to investigate the activation of the TGF-β and BMP signaling pathways. The kit contains enough primary and secondary antibodies to perform four Western blot experiments per 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

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 9; 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-SMADs dissociate from the receptor and form a heteromeric complex with SMAD4, initiating translocation of the heteromeric SMAD complex to the nucleus. Once in the nucleus, SMADs recruit a variety of DNA binding proteins that function to regulate transcriptional activity (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. Wrana, J.L. (2000) Sci STKE 2000, re1.
  7. Attisano, L. and Wrana, J.L. (2002) Science 296, 1646-7.
  8. Moustakas, A. et al. (2001) J Cell Sci 114, 4359-69.

Background References

    Trademarks and Patents

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

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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 1
    #9963

    Phospho-Smad Antibody Sampler Kit

    Phospho-Smad Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1.0 mg/mL) from serum starved HT-1080 cells treated with hTGF-β3 (10 ng/mL, 30 min) using Phospho-SMAD2 (Ser465/467) (138D4) Rabbit mAb #3108. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (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.
    Phospho-Smad 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.
    Phospho-Smad Antibody Sampler Kit: Image 3 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from serum-starved HT1080 cells treated with hTGF-beta3 (10 ng/mL, 30 min) using Phospho-SMAD3 (Ser423/425) (C25A9) Rabbit mAb #9520. The virtual lane view (left) shows the target band (as indicated) at a 1:250 dilution of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at a 1:250 dilution (gray line) 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.
    Phospho-Smad Antibody Sampler Kit: Image 4 Expand Image
    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).
    Phospho-Smad Antibody Sampler Kit: Image 5 Expand Image
    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.
    Phospho-Smad Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using SMAD1 (D59D7) XP® Rabbit mAb.
    Phospho-Smad Antibody Sampler Kit: Image 7 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.
    Phospho-Smad Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from COS, NIH3T3, PC12, and SK-N-MC cells, using Smad4 Antibody.
    Phospho-Smad 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.
    Phospho-Smad Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from SK-N-MC, COS and PC12 cells, using SMAD5 Antibody.
    Phospho-Smad Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from various cell lines using Smad6 Antibody.
    Phospho-Smad Antibody Sampler Kit: Image 12 Expand Image
    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 #9520 (upper) or total SMAD3 (C67H9) Rabbit mAb #9523 (lower).
    Phospho-Smad Antibody Sampler Kit: Image 13 Expand Image
    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.
    Phospho-Smad Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from various cell lines using Smad2 (D43B4) XP® Rabbit mAb.
    Phospho-Smad Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from NIH/3T3 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Smad4 siRNA I (Mouse Specific) (+), using Smad4 Antibody #9515 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The Smad4 Antibody confirms silencing of Smad4 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
    Phospho-Smad Antibody Sampler Kit: Image 16 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).
    Phospho-Smad Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of extracts from HT1080 (human), C2C12 (mouse) and B35 (rat) using SMAD3 (C67H9) Rabbit mAb.
    Phospho-Smad Antibody Sampler Kit: Image 18 Expand Image
    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).
    Phospho-Smad Antibody Sampler Kit: Image 19 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HaCaT cells treated with Human TGF-β3 #3706 (7ng/ml) for 1 h and either 20 μl of Smad4 Antibody or 2 μl of 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.
    Phospho-Smad Antibody Sampler Kit: Image 20 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).
    Phospho-Smad Antibody Sampler Kit: Image 21 Expand Image
    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 #9520 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.
    Phospho-Smad Antibody Sampler Kit: Image 22 Expand Image
    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).
    Phospho-Smad Antibody Sampler Kit: Image 23 Expand Image
    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.
    Phospho-Smad Antibody Sampler Kit: Image 24 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.
    Phospho-Smad Antibody Sampler Kit: Image 25 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.
    Phospho-Smad Antibody Sampler Kit: Image 26 Expand Image
    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).
    Phospho-Smad Antibody Sampler Kit: Image 27 Expand Image
    Flow cytometric analysis of HT-1080 cells using SMAD3 (C67H9) Rabbit mAb #9523 (blue) compared to a nonspecific negative control antibody (red).
    Phospho-Smad Antibody Sampler Kit: Image 28 Expand Image
    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.
    Phospho-Smad Antibody Sampler Kit: Image 29 Expand Image
    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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across CDKN1A, a known target gene of SMAD3 (see additional figure containing ChIP-qPCR data).
    Phospho-Smad Antibody Sampler Kit: Image 30 Expand Image
    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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #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).
    Phospho-Smad Antibody Sampler Kit: Image 31 Expand Image
    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® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. 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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    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.