重组Anti-ATF2 (phospho T71)抗体[E268]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

Immunocytochemistry of HeLa (Human epithelial cell line from cervix adenocarcinoma), prepared in FBS free medium overnight labeling ATF2 at 0.9 μg/ml. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. AlexaFluor^®488 Goat anti-Rabbit (ab150077) was used as the secondary antibody at 1/500 . Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as the counter stain at 2.5 μg/ml. DAPI was used for nuclear counter stain. Confocal image showing the expression was increased on HeLa cells, prepared in FBS free medium overnight, then treated with 250ng/ml anisomycin for 30min.

• IP

Supplier Data

Immunoprecipitation - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

ab32019 Immunoprecipitating ATF2 in Human Hela whole cell lysate. For western blotting ab32019 (1 : 1000) was used to confirm successful immunoprecipitation. Blocking and diluting buffer used was 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-ATF2 (phospho T71) antibody [E268] (ab32019) at 0.47 µg/mL

Lane 1:

HeLa (human cervix adenocarcinoma) treated with 250ng/ml anisomycin for 30min whole cell lysate (input) at 10 µg

Lane 2:

HeLa treated with 250ng/ml anisomycin for 30min whole cell lysate (+)

Lane 3:

Rabbit monoclonal IgG (<a href='/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab32019 in HeLa treated with 250ng/ml anisomycin for 30min whole cell lysate (-)

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/10000 dilution

Predicted band size: 55 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

Blocking and diluting buffer used was 2% BSA/TBST .
The molecular weight is 70kD, consistent with the literature (PMID : 24223142).

All lanes:

Western blot - Anti-ATF2 (phospho T71) antibody [E268] (ab32019) at 0.1 µg/mL

Lane 1:

HeLa (human cervix adenocarcinoma), prepared in FBS free medium overnight, whole cell lysate at 10 µg

Lane 2:

HeLa, prepared in FBS free medium overnight, then treated with 250ng/ml anisomycin for 30 minutes whole cell lysate at 10 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 1/100000 dilution

Predicted band size: 55 kDa

Observed band size: 70 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

Blocking/Diluting Buffer and concentration : 5% NFDM /TBST

All lanes:

Western blot - Anti-ATF2 (phospho T71) antibody [E268] (ab32019) at 0.526 µg/mL

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) grown in serum free media for overnight, whole cell lysate at 20 µg

Lane 2:

HeLa grown in serum free media for overnight, then treated with 250ng/ml Anisomycin for 30min, whole cell lysate at 20 µg

Lane 3:

HeLa grown in serum free media for overnight, then treated with 250ng/ml Anisomycin for 30min whole cell lysate, then the membrane was incubated with alkaline phosphatase at 20 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 0.05 µg/mL

Predicted band size: 55 kDa

Observed band size: 70 kDa

false

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ K-562 (human chronic myelogenous leukemia lymphoblast) cells treated with anisomycin (25 µg/mL 30 min) and 5 µg of ab ab32019[E268]. 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.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ K-562 (human chronic myelogenous leukemia lymphoblast) cells treated with anisomycin (25 µg/mL 30 min) and 5 µg of ab ab32019[E268]. 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.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ K-562 (human chronic myelogenous leukemia lymphoblast) cells treated with anisomycin (25 µg/mL 30 min) and 5 µg of ab ab32019[E268]. 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.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

Blocking and diluting buffer used was 2% BSA/TBST.

All lanes:

Western blot - Anti-ATF2 (phospho T71) antibody [E268] (ab32019) at 0.1 µg/mL

Lane 1:

Untreated NIH/3T3 (mouse embryo), prepared in FBS free medium overnight, whole cell lysate at 10 µg

Lane 2:

NIH/3T3, prepared in FBS free medium overnight, then treated with 10 μg/ml anisomycin for 30 minutes whole cell lysate at 10 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 1/100000 dilution

Predicted band size: 55 kDa

Observed band size: 70 kDa

false

Exposure time: 5s

• Dot

Lab

Dot Blot - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

Dot blot analysis of ATF2 (phospho T71) peptide (Lane 1) and ATF2 non-phospho peptide (Lane 2) using ab32019 at 1/1000 dilution followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution.

Exposure time : 3 minutes

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

• WB

CiteAb

Western blot - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

Western Blotting using Anti-ATF2 (phospho T71) antibody [E268], ab32019. Publication image from Sun, B. et al., 2016, Nat Commun, 27853137. Legend direct from paper.

TGF-β increases CUGBP1 expression in HSCs via p38 MAPK.(a) Quantitative PCR analyses of CUGBP1 mRNA from the human hepatic stellate cell line LX-2 or human hepatocyte L02 cells treated with or without 5 ng ml−1 TGF-β for 6 h (mean±s.e.m.; n=3, **P<0.01 by Student's t-test). (b,c) Western blot analyses of CUGBP1 from LX-2 cells, primary mouse HSCs (b), and primary mouse hepatocytes (c) treated with or without 5 ng ml−1 TGF-β for 24 h. The data are representative of three independent experiments (mean±s.e.m.; n=3, **P<0.01 by Student's t-test). (d,e) LX-2 cells were treated with actinomycin D (1 µg ml−1) and with or without 5 ng ml−1 TGF-β for indicated time intervals (d). LX-2 cells were treated with or without SB431542 (10 µM), SB203580 (10 µM), SP600125 (10 µM), FR180204 (10 µM) or SIS3 (20 µM), following 5 ng ml−1 TGF-β treatment for 6 h (e). And then quantitative PCR was carried out to detect the remaining mRNA expression of CUGBP1. (mean±s.e.m.; n=3, *P<0.05, **P<0.01 by one-way analysis of variance followed by Dunnett's test). (f) Western blot analyses of LX-2 cells treated with or without SB431542, following 5 ng ml−1 TGF-β treatment for 24 h. (g) Gene2promotor analyses of promoter and transcription factors of human CUGBP1 gene. (h) Probe pull down assay was performed by mixing CUGBP1-CRE-Bio or mCUGBP1-CRE-Bio with total cell extracts from LX-2 cells treated as in f. Precipitates were prepared for Western blotting using SoftLink Soft Release avidin resin. The data in f and h are representative of two independent experiments.

false

• WB

CiteAb

Western blot - Anti-ATF2 (phospho T71) antibody [E268] (AB32019)

Western Blotting using Anti-ATF2 (phospho T71) antibody [E268], ab32019. Publication image from Sun, B. et al., 2016, Nat Commun, 27853137. Legend direct from paper.

TGF-β increases CUGBP1 expression in HSCs via p38 MAPK.(a) Quantitative PCR analyses of CUGBP1 mRNA from the human hepatic stellate cell line LX-2 or human hepatocyte L02 cells treated with or without 5 ng ml−1 TGF-β for 6 h (mean±s.e.m.; n=3, **P<0.01 by Student's t-test). (b,c) Western blot analyses of CUGBP1 from LX-2 cells, primary mouse HSCs (b), and primary mouse hepatocytes (c) treated with or without 5 ng ml−1 TGF-β for 24 h. The data are representative of three independent experiments (mean±s.e.m.; n=3, **P<0.01 by Student's t-test). (d,e) LX-2 cells were treated with actinomycin D (1 µg ml−1) and with or without 5 ng ml−1 TGF-β for indicated time intervals (d). LX-2 cells were treated with or without SB431542 (10 µM), SB203580 (10 µM), SP600125 (10 µM), FR180204 (10 µM) or SIS3 (20 µM), following 5 ng ml−1 TGF-β treatment for 6 h (e). And then quantitative PCR was carried out to detect the remaining mRNA expression of CUGBP1. (mean±s.e.m.; n=3, *P<0.05, **P<0.01 by one-way analysis of variance followed by Dunnett's test). (f) Western blot analyses of LX-2 cells treated with or without SB431542, following 5 ng ml−1 TGF-β treatment for 24 h. (g) Gene2promotor analyses of promoter and transcription factors of human CUGBP1 gene. (h) Probe pull down assay was performed by mixing CUGBP1-CRE-Bio or mCUGBP1-CRE-Bio with total cell extracts from LX-2 cells treated as in f. Precipitates were prepared for Western blotting using SoftLink Soft Release avidin resin. The data in f and h are representative of two independent experiments.

false