Recombinant Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434)

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).

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.

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.

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.

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.

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).

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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.