Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Smad2 + Smad3 with ab202445 at 1/600 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Smad2 + Smad3 with ab202445 at 1/200 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). The results show signal translocation after TGF-beta (10ng/ml, 1h) treatment on HeLa cells.The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab202445 at 1/200 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution,followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• ChIP

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ChIP - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

Chromatin was prepared from HaCaT (Human keratinocyte cell line) cells treated with 7ng/ml TGF-β for 1h and non-treated according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab202445 (blue), and 20μl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

• IP

Supplier Data

Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

Smad2 + Smad3 was immunoprecipitated from 0.35mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab202445 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab202445 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10μg (Input).

Lane 2 : ab202445 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab202445 in HeLa whole cell lysate.

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

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445)

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• WB

Supplier Data

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 30 seconds; Lane 2 : 10 seconds.

All lanes:

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/2000 dilution

Lane 1:

Human fetal heart lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 52 kDa

Observed band size: 58-62 kDa

false

• WB

Supplier Data

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2 : 10 seconds; Lane 3 : 3 seconds; Lane 4 : 1 seconds.

All lanes:

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/2000 dilution

Lane 1:

293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 2:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 10 µg

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 58-62 kDa

false

• WB

Supplier Data

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

Blocking/Dilution buffer : 5% NFDM/TBST.

Human Smad2 fragment recombinant protein contains aa2-270 with His-Tag®. Human Smad3 fragment recombinant protein contains aa2-227 with His-Tag® and GST-tag.

All lanes:

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/1000 dilution

Lane 1:

Recombinant protein fragment human Smad2 at 0.01 µg

Lane 2:

Recombinant protein fragment human Smad3 at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 38 kDa,60 kDa

false

Exposure time: 1s

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

ChIC/CUT&RUN was performed using ta pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HaCaT (Human keratinocyte cell line) cells (treated with 7ng/ml TGF-β for 1h) and 5 µg of ab202445 [EPR19557-4]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Supplier Data

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 3 minutes; Lane 2 : 10 seconds; Lane 3 : 30 seconds; Lane 4 : 3 seconds; Lane 5 : 5 seconds.

Lane 1:

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/1000 dilution

Lanes 2 and 5:

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/2000 dilution

Lanes 3 - 4:

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/20000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Rat brain lysate at 10 µg

Lane 4:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 5:

Rat spleen lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 58-62 kDa

false

• WB

CiteAb

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (AB202445)

Smad2 + Smad3 western blot using anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade ab202445. Publication image and figure legend from Song, Y., Wang, Z., et al., 2020, J Cell Mol Med, PubMed 33124760.

ab202445 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 ab202445 please see the product overview.

Effects of DEK shRNA on Smad, MAPK and PI3K/AKT signalling pathways in BEAS-2B cells. (A-E) The expressions of TGF-β, DEK mad2/3 and Smad4, ERK1/2, p38, JNK, and PI3K, AKT, mTOR were detected by Western blot. The relative density of each protein was calculated. Data were shown as mean ± SD (n = 3). NC, negative control cells; ovDEK, cells transfected with DEK; shNC, cells transfected with scrambled shRNA; shDEK, cells transfected with DEK shRNA. *p < 0.05, vs control group. #p < 0.05, ovDEK vs TGF-β1 + NC group; shDEK vs TGF-β1 + shNC group

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