Revision 2

#11958

Store at -20C

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
SMAD2 (D43B4) XP® Rabbit mAb5339100 µl60 kDaRabbit IgG
Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb18338100 µl60 kDaRabbit IgG

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

UniProt ID:
#Q15796, #Q62432

Entrez-Gene Id:
4087, 17126

Description

PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.

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).

Trademarks and Patents

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

PhosphoPlus 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.

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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.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from HaCaT cells, untreated (-) or treated with Human Transforming Growth Factor β3 (hTGF-β3) #8425 (100ng/ml, 30mins) (+), using Phospho SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb (upper) and total Smad2 (D43B4) XP® Rabbit mAb, #5339 (lower).
undefined Image 1: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
Western blot analysis of extracts from HT-1080 cells, untreated (-) or treated with hTGF-β3 #8425 (10 ng/ml, 30 min; +), using Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb #18338 (Panel A) and SMAD2 (L16D3) Mouse mAb #3103 (Panel B). Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) #5151 (green) and Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470 (red) were used as secondary antibodies.
undefined Image 2: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
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.
undefined Image 3: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
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

Cell Signaling Technology Logo
Immunoprecipitation of phospho-SMAD2 (Ser465/Ser467) from extracts of HaCaT cells treated with Human Transforming Growth Factor β1 (hTGF-β1) #8915 (10nM, 30mins). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb. Western blot analysis was performed using P-SMAD2 (Ser465/ Ser467) (E8F3R) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used for detection to avoid cross-reactivity with IgG.
undefined Image 4: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
Western blot analysis of extracts from various cell lines using Smad2 (D43B4) XP® Rabbit mAb.
undefined Image 5: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
Confocal immunofluorescent analysis of serum-starved HT-1080 cells, untreated (left), treated with Human Transforming Growth Factor β3 (hTGF-β3) #8425 (100 ng/mL, 20 min; center), or treated with hTGF-β3 and post-processed with λ-phosphatase (right), using Phospho-Smad2 (Ser465/467) (E8F3R) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).
undefined Image 6: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
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

Cell Signaling Technology Logo
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).
undefined Image 7: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
Flow cytometric analysis of serum-starved HT-1080 cells, untreated (blue), treated with Human Transforming Growth Factor β3 (hTGF-β3) #8425 (100 ng/mL, 30 min; green) or pretreated with SB43152 (10 ug/mL, 30 min) and treated with hTGF-β3 (100 ng/mL, 30 min; red), using Phospho-Smad2 (Ser465/467) (E8F3R) 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.
undefined Image 8: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
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.
undefined Image 9: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
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

Cell Signaling Technology Logo
Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with Human Growth Factor β1 #8915 (7 ng/ml, 1 hr) and either Phospho-SMAD2 (Ser465/Ser467) (E8F3R) 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 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, which is equivalent to one.
undefined Image 10: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
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® 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.
undefined Image 11: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
Immunoprecipitation of Smad2 from HeLa cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is SMAD2 (D43B4) XP® Rabbit mAb. Western blot analysis was performed using SMAD2 (L16D3) Mouse mAb #3103. Anti-mouse IgG, HRP-linked Antibody #7076 was used as a secondary antibody.
undefined Image 12: PhosphoPlus<sup>®</sup> SMAD2 (Ser465/467) Antibody Duet
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.