All lanes : Anti-ATF1 antibody [EPR17028] (ab181569) at 1/1000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Rat heart lysate
Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 29 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:10574952).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling ATF1 with ab181569 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 nuclear staining on HeLa cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling ATF1 with ab181569 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on mouse liver (PMID: 11865068; PMID: 28032861). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

ATF1 was immunoprecipitated from 1 mg of mouse kidney lysate with ab181569 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab181569 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Mouse kidney lysate 10 µg (Input). 

Lane 2: ab181569 IP in mouse kidney lysate (+). 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab181569 in mouse kidney lysate (-).

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

Exposure time: 30 seconds.

Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling ATF1 with ab181569 at 1/150 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-ATF1 antibody [EPR17028] (ab181569) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) 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

Developed using the ECL technique.

Predicted band size: 29 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?

 

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

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-ATF1 antibody [EPR17028] (ab181569) at 1/1000 dilution

Lane 1 : Mouse kidney lysate
Lane 2 : Mouse kidney lysate treated with alkaline phosphatase for 1 hour at 37°C

Lysates/proteins at 2.5 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 29 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

ATF1 has been shown to be phosphorylated in cultured cells and hyperphosphorylation in tissues (PMID:20730097). Treatment of cell lysates with alkaline phosphatase lead to complete dephosphorylation, while we were only able to partial dephosphorylate ATF1 in tissue lysates.

 

All lanes : Anti-ATF1 antibody [EPR17028] (ab181569) at 1/1000 dilution

Lane 1 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate treated with alkaline phosphatase for 1 hour at 37°C

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Developed using the ECL technique.

Predicted band size: 29 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

ATF1 has been shown to be phosphorylated in cultured cells and hyperphosphorylation in tissues (PMID:20730097). Treatment of cell lysates with alkaline phosphatase lead to complete dephosphorylation, while we were only able to partial dephosphorylate ATF1 in tissue lysates.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling ATF1 with ab181569 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 nuclear staining on NIH/3T3 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.