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77397
TGF-β Fibrosis Pathway Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

TGF-β Fibrosis Pathway Antibody Sampler Kit #77397

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TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 1

Confocal immunofluorescent analysis of mouse small intestine (left) and skeletal muscle (right) using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 2

Immunohistochemical analysis of paraffin-embedded human endometrioid carcinoma using alpha-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb performed on the Leica® Bond™ Rx.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 3

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 4

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 5

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 6

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 7

Confocal immunofluorescent analysis of THP-1 cells (left, positive) and A-204 cells (right, negative) using YKL-40 (E2L1M) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 8

Western blot analysis of extracts from HaCaT cells, untreated (-) or treated with hTGF-β3 #8425 (+) in the absence or presence of the TGFR inhibitor SB 431542, using Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (upper) or Smad2/3 (D7G7) XP® Rabbit mAb #8685 (lower).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 9

Western blot analysis of recombinant active TGF-β1 using TGF-β (56E4) Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 10

Western blot analysis of extracts from Hep G2 cells, untreated (-) or treated with PNGase F (+), and untreated A172 cells, using TGF-β Receptor II Antibody (upper), and β-Actin (D6A8) Rabbit mAb #8457 (lower).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 11

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 12

Immunoprecipitation of COL1A1 protein from U-118 MG cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is COL1A1 (E8I9Z) XP® Rabbit mAb. Western blot analysis was performed using COL1A1 (E8I9Z) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as secondary antibody.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 13

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 14

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 15

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 16

Immunohistochemical analysis of paraffin-embedded human lung squamous cell carcinoma using YKL-40 (E2L1M) Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 17

Western blot analysis of extracts of HeLa cells, mock transfected or transfected with TGF-β1 precursor, using TGF-β (56E4) Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 18

Western blot analysis of extracts from 293T cells, mock-transfected (-) or transfected with a plasmid expression construct encoding myc/DDK-tagged human TGF-β Receptor II (+), using TGF-β Receptor II Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 19

Immunohistochemical analysis of paraffin-embedded human appendix using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 20

Western blot analysis of extracts from various cell lines using COL1A1 (E8I9Z) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 21

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 22

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).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 23

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).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 24

Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's lymphoma using YKL-40 (E2L1M) Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 25

Western blot analysis of extracts from K-562, Saos-2 and 786-0 cells using TGF-β (56E4) Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 26

Immunohistochemical analysis of paraffin-embedded human ductal carcinoma of the breast using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 27

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).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 28

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 29

Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using YKL-40 (E2L1M) Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 30

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 31

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.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 32

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

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 33

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).

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 34

Immunohistochemical analysis of paraffin-embedded THP-1 cell pellet (left, positive) or A-204 cell pellet (right, negative) using YKL-40 (E2L1M) Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 35

Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 36

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

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 37

Western blot analysis of extracts from THP-1 and A-204 using YKL-40 (E2L1M) Rabbit mAb upper) and loading control α-Actinin (D6F6) XP® Rabbit mAb #6487. As expected, A-204 is low to negative for YKL-40 expression.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 38

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 39

Immunohistochemical analysis of paraffin-embedded mouse skeletal muscle using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 40

Immunohistochemical analysis of paraffin-embedded mouse small intestine using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 41

Immunoprecipitation of α-smooth muscle actin from mouse colon tissue extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb. Western blot analysis was performed using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb.

TGF-β Fibrosis Pathway Antibody Sampler Kit: Image 42

Western blotting analysis of extracts from various human, mouse, and rat tissues using α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb (upper) and GAPDH #5174 (lower). As expected, skeletal muscle samples are negative for α-smooth muscle actin.

To Purchase # 77397T
Product # Size Price Inventory
77397T
1 Kit  (9 x 20 µl)

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb 19245 20 µl
  • WB
  • IP
  • IHC
  • IF
H M R 42 Rabbit IgG
COL1A1 (E8I9Z) Rabbit mAb 91144 20 µl
  • WB
  • IP
H M R 220 Rabbit IgG
Smad2/3 (D7G7) XP® Rabbit mAb 8685 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H Mk M R 52, 60 Rabbit IgG
Smad2 (D43B4) XP® Rabbit mAb 5339 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H Mk M R 60 Rabbit IgG
Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb 18338 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R 60 Rabbit IgG
YKL-40 (E2L1M) Rabbit mAb 47066 20 µl
  • WB
  • IHC
  • IF
H 30-40 Rabbit IgG
Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb 8828 20 µl
  • WB
H Mk M R 52, 60 Rabbit IgG
TGF-β (56E4) Rabbit mAb 3709 20 µl
  • WB
H 12, 45-60 Rabbit 
TGF-β Receptor II Antibody 79424 20 µl
  • WB
H M R 85 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The TGF-β Fibrosis Pathway Antibody Sampler Kit provides an economical means of investigating activation of TGF-β/ SMAD2/3 signaling pathways in cells or tissues that lead to the expression of profibrotic genes, including expression of α-Smooth Muscle Actin in activated fibroblasts, and upregulation of Collagen1A1, Col11A1, and YKL-40. The kit includes enough antibodies to perform at least two western blot experiments with each primary antibody.

Specificity / Sensitivity

α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb recognizes endogenous levels of total α-smooth muscle protein. COL1A1 (E8I9Z) Rabbit mAb recognizes endogenous levels of total COL1A1 protein. Smad2/3 (D7G7) XP® Rabbit mAb recognizes endogenous levels of total Smad2/3 protein. Smad2 (D43B4) XP® Rabbit mAb detects endogenous levels of total Smad2 protein. This antibody does not cross-react with Smad3. Phospho-Smad2 (Ser465/Ser467) (E8F3R) Rabbit mAb recognizes endogenous levels of Smad2 protein when phosphorylated at Ser465 and Ser467. YKL-40 (E2L1M) Rabbit mAb recognizes endogenous levels of total YKL-40 protein. Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb recognizes endogenous levels of Smad2 protein when phosphorylated at Ser465 and Ser467. This antibody also recognizes endogenous levels of Smad3 protein when phosphorylated at Ser422 only or at both Ser423 and Ser425. TGF-β (56E4) Rabbit mAb detects recombinant TGF-β1 and TGF-β3 proteins. The antibody also detects endogenous levels of the TGF-β precursor proteins. TGF-β Receptor II Antibody recognizes endogenous levels of total TGF-β Receptor II protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human α-Smooth Muscle Actin protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Phe1197 of human COL1A1 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His198 of human Smad2/3 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of mouse Smad2 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser465/467 of human Smad2 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human YKL-40 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser465/467 of human Smad2 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to a region in the carboxy terminus of TGF-β1 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp247 of human TGF-β Receptor II protein. Antibodies are purified by protein A and peptide affinity chromatography.

Background

Transforming growth factor-β (TGF-β) superfamily members are critical regulators of cell proliferation and differentiation, developmental patterning and morphogenesis, and disease pathogenesis (1-4). In the context of fibrosis, TGF-β signaling to SMAD2/3 is one of the biggest drivers of the profibrotic program (5).

TGF-β elicits signaling through three cell surface receptors: type I (RI), type II (RII), and type III (RIII). In response to ligand binding, the type II receptors form stable heterotrimeric complexes with the type I receptors, allowing phosphorylation and activation of type I receptor kinase. Activated type I receptors associate with SMAD2/3 and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated SMADs dissociate from the receptor and form a heterotrimeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, phosphorylated SMAD2/3 targets a subset of DNA binding proteins to regulate the transcriptional program (6-8).

In the context of fibrosis, SMAD2/3 activation upregulates expression of profibrotic genes such as COL1A1 and other ECM modulators that modify the extracellular matrix of the tissue. (9). TGF-β/ SMAD2/3 signaling also induces expression of α-Smooth Muscle Actin in fibroblasts, causing transformation of these cells to myofibroblasts (10). Myofibroblasts further modify the ECM, causing excessive accumulation of collagens and other ECM components. Injury to the tissue attracts macrophages and other immune cells and the fibrotic tissue soon becomes a site of inflammation (11). In this pro-fibrotic, pro-inflammatory environment, YKL-40, also known as Chitinase-3-like protein 1 (CHI3L1), is secreted. YKL-40 is a pro-inflammatory glycoprotein that also contributes to the progression of fibrosis (12). Measurement of collagen content, α-Smooth Muscle Actin, and the release of YKL-40 are predictive of fibrotic activity.

  1. Massague, J. et al. (2000) Cell 103, 295-309.
  2. Caestecker, M.P. et al. (2000) J. Natl. Cancer Inst. 92, 1388-1402.
  3. Derynck, R. et al. (2001) Nature Genet. 29, 117-129.
  4. Miyazono, K. et al. (2000) Adv. Immunol. 75, 115-157.
  5. Meng, X.M. et al. (2016) Nat Rev Nephrol 12, 325-38.
  6. Wu, G. et al. (2000) Science 287, 92-7.
  7. Attisano, L. and Wrana, J.L. (2002) Science 296, 1646-7.
  8. Moustakas, A. et al. (2001) J Cell Sci 114, 4359-69.
  9. Bagalad, B.S. et al. J Oral Maxillofac Pathol 21, 462-3.
  10. Mack, M. (2018) Matrix Biol 68-69, 106-21.
  11. Johansen, J.S. (2006) Dan Med Bull 53, 172-209.

Pathways & Proteins

Explore pathways + proteins related to this product.

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For Research Use Only. Not For Use In Diagnostic Procedures.
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XP is a registered trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 5,675,063.