All lanes : Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ATF3 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 21 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab207434 observed at 21 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab207434 was shown to react with ATF3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264908 (knockout cell lysate ab257073) was used. Wild-type HeLa and ATF3 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab207434 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab207434 staining ATF3 in wild-type Hap1 cells (top panel) and ATF3 knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab207434 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

All lanes : Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : ATF3 knockout HAP1 whole cell lysate
Lane 3 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 21 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab207434 observed at 21 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab207434 was shown to specifically react with ATF3 in wild-type HAP1 cells as signal was lost in ATF3 knockout cells. Wild-type and ATF3 knockout samples were subjected to SDS-PAGE. ab207434 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling ATF3 with ab207434 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased nuclear staining on RAW 264.7 cell line, after treatment with LPS (1µg/ml) for 2 hours.

The nuclear counter stain is DAPI (blue). Tubulin is detected with 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: ab207434 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

All lanes : Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : ATF3 knockout HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 21 kDa

Lanes 1-2: Merged signal (red and green). Green - ab207434 observed at 21 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab207434 Anti-ATF3 antibody [EPR19488] - ChIP Grade was shown to specifically react with ATF3 in wild-type HCT116 cells. Loss of signal was observed when knockout cell line ab266872 (knockout cell lysate ab257074) was used. Wild-type and ATF3 knockout samples were subjected to SDS-PAGE. ab207434 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : ATF3 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 21 kDa

Lanes 1-2: Merged signal (red and green). Green - ab207434 observed at 21 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab207434 Anti-ATF3 antibody [EPR19488] - ChIP Grade was shown to specifically react with ATF3 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab266955 (knockout cell lysate ab257075) was used. Wild-type and ATF3 knockout samples were subjected to SDS-PAGE. ab207434 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Chromatin was prepared from Hela cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab207434 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

All lanes : Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434) at 1/500 dilution

Lane 1 : 293T (Human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : Human liver tissue lysate
Lane 3 : Raw264.7 (Mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 4 : Mouse liver tissue lysate
Lane 5 : MEF (Mouse embryonic fibroblast (immortalized)) whole cell lysate
Lane 6 : Mouse heart tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 21 kDa
Observed band size: 21 kDa


Exposure time: 180 seconds

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

ATF3 has a low expression level in some cell lines and tissues, but is increased under treatment (PMID: 8622660, PMID: 22053207, PMID: 20018623, PMID: 29940414).

Rabbit monoclonal [EPR16891] to GAPDH (ab181602) used as loading control.

All lanes : Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434) at 1/1000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lane 4 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate
Lane 5 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 6 : LnCap (Human prostate carcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 21 kDa
Observed band size: 21,23 kDa why is the actual band size different from the predicted?


Exposure time: 180 seconds

Blocking/Diluting buffer and concentration 5% NFDM /TBST

ATF3 migrates as a 21 and 23 kDa doublet band due to an alternative ATG usage (PMID: 12225289, PMID: 8649793)

The mRNA and protein expression of ATF3 is low or undetectable in most cells, but its expression is rapidly induced by a large variety of cellular stresses including DNA damage, wounds, and cellular injury (PMID: 19136462, 20651982, 20592017).

All lanes : Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434) at 1/1000 dilution

Lane 1 : Untreated THP-1 (Human monocytic leukemia cell line) whole cell lysate
Lane 2 : THP-1 (Human monocytic leukemia cell line) treated with 80nM TPA overnight, then treated with 1 µg/ml LPS for 8 hours whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 21 kDa
Observed band size: 21 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID: 24973221).

All lanes : Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434) at 1/1000 dilution

Lane 1 : Untreated RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) treated with 1 µg/ml LPS for 2 hours whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 21 kDa
Observed band size: 21 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID: 24973221, PMID: 19136462).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (Human monocytic leukemia cell line) cells labeling ATF3 with ab207434 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased nuclear staining on THP-1 cell line, after treatment with TPA (80nM) for overnight, followed by LPS (1µg/ml) for 8 hours.

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: ab207434 at 1/100 dilution followed by 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.

ab207434 at 1/50 immunoprecipitating ATF3 in HeLa (human cervix adenocarcinoma) cells.

Lane 1 (input): HeLa whole cell lysate 10μg
Lane 2 (+): ab207434 + HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab207434 in HeLa (human cervix adenocarcinoma) whole cell lysate

For western blotting, ab207434 (1:500) as primary antibody and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

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