重组Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467)抗体[EP728(2)AY]

• WB

Lab

Western blot - Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] (AB76296)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] (ab76296) at 1/1000 dilution

Lane 1:

Untreated HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

HeLa treated with 20ng/ml TGF beta 1 for 15 min whole cell lysate at 15 µg

Lane 3:

HeLa treated with 20ng/ml TGF beta 1 for 15 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 52 kDa

false

Exposure time: 20s

• WB

Unknown

Western blot - Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] (AB76296)

All lanes:

Western blot - Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] (ab76296) at 1/500 dilution

Lane 1:

HeLa cell lysate - untreated at 10 µg

Lane 2:

HeLa cell lysate - treated with BMP-4 at 10 µg

Secondary

All lanes:

goat anti-rabbit HRP at 1/2000 dilution

Observed band size: 52 kDa

false

• WB

Lab

Western blot - Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] (AB76296)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] (ab76296) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 15 µg

Lane 2:

NIH/3T3 treated with 20ng/ml TGF beta 1 and 50µM MG-132 for 15 min whole cell lysate at 15 µg

Lane 3:

NIH/3T3 treated with 20ng/ml TGF beta 1 and 50µM MG-132 for 15 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour at 15 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 52 kDa

false

Exposure time: 100s

• WB

Lab

Western blot - Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] (AB76296)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] (ab76296) at 1/1000 dilution

Lane 1:

Untreated C6 (Rat glial tumor glial cell) whole cell lysate at 15 µg

Lane 2:

C6 treated with 10ng/ml TGF beta 3 and 50µM MG-132 for 30 min whole cell lysate at 15 µg

Lane 3:

C6 treated with 10ng/ml TGF beta 3 and 50µM MG-132 for 30 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 52 kDa

false

Exposure time: 7s

• WB

CiteAb

Western blot - Anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] (AB76296)

SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) western blot using anti-SMAD1 + SMAD5 + SMAD9 (phospho S463 + S465 + S467) antibody [EP728(2)AY] ab76296. Publication image and figure legend from Ghiabi, P., Jiang, J., et al., 2015, J Transl Med, PubMed 25623554.

ab76296 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab76296 please see the product overview.

Mesenchymal phenotypes in ECsMesis regulated by synergistic Notch and TGFβ pathways. A) Semi-quantitative qPCR showing the over-expression of notch transcription factors in sorted ECsMes. (*** p < 0.001, ** p < 0.01, mean ± SEM, n = 3). B) Western analysis showing stable p-Smad3 expression in ECsNorm and ECsMes compared to total protein. C) Western analysis demonstrating the activation of Smad5 in ECsMes compared to ECsNorm (top lane). Once ECsNorm were treated with Jag1 and/or TGFβ1, p-Smad5 was up-regulated (top lane). Hes-1 up-regulation in response to TGFβ1 or Jag1 treatments suggests notch pathway involvement (third lane). Simultaneous supplementation of ligands triggered dramatic Hes-1 up-regulation in ECsNorm implying a synergistic effect. D) Western analysis displaying p-Smad1/5 down-regulation in response to notch and/or TGFβ inhibition. GSI and SB were added to ECsNorm-MDA-231 co-cultures, then ECs were sorted for analyzing p-Smad1/5 down-regulation comparing to total protein (left panel). Inhibition also down-regulated Hes-1 activation (right panel). Concurrent inhibitions of ECsMes dramatically reduced p-Smad1/5 and Hes-1 activation. E) Confocal images demonstrate endothelial and mesenchymal states of ECs co-cultured with MDA-231Jag1-KD or MDA-231Scrambled. ECs grown with MDA-231Jag1-KD demonstrated normal endothelial feature (Upper panels). However, they displayed decreased mesenchymal phenotype compared to ECsMes (sorted from MdA-231Scrambled) (bottom panel). Scales are 10 μm. F) Semi-quantitative qPCR compared expression of endothelial and mesenchymal markers in ECsMes and ECs sorted from MDA-231Jag1-KD cells. Silencing Jag1 and notch down-regulation in MDA-231Jag1-KD counteracted the acquisition of mesenchymal phenotype by ECs. G) Wound healing assay to assess the migratory capacity of ECs sorted from MDA-231Jag1-KD (ECs St. MDA-231Jag1-KD) and ECMes (sorted from MDA-231Scrambled) (left panels). After sorting, a 48-hour wound healing assay was initiated under complete starvation to exclude the possibility of cell proliferation. The invasion property of ECs St. MDA-231Jag1-KD decreased by 1.6-fold compared to ECsMes (right panel).

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