Image 1: Crystal structure of ATF3 (Trg-Tpl Seq Id 50.75%).

ATF3 Target Introduction

Protein Function

• The protein ATF-3 (Cyclic AMP-dependent transcription factor ATF-3) is a transcription factor that binds to cAMP response elements (CRE), which are sequences present in many viral and cellular promoters.
• ATF-3 exists in 5 isoforms. Isoform 1: inhibits transcription with ATF site promoters, possibly by stabilizing the inhibition of auxiliary factors binding to the promoter. Isoform 2: activates transcription, possibly by separating inhibitory auxiliary factors from the promoter to activate transcription. Isoform 3: stress-induced isoform, counteracts the transcriptional inhibition of isoform 1.

Protein Expression

• ATF3 is expressed at low levels in some cell lines and tissues (such as MCF7, MDA-MB-231, MEF; liver, heart), but its expression levels increase after stimulation.

Protein Localization

• Localized in the cell nucleus.

Figure 2: Experimental results of ATF3, Immunocytochemistry/Immunofluorescence - Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434). Green: ATF3; Red: alpha Tubulin; Blue: DAPI. The confocal image shows enhanced nuclear staining of RAW 264.7 cell line after treatment with LPS (1 µg/ml) for 2 hours.

Isoforms & post-translational modifications

• Human (P18847): Isoform 1, 20 kDa, Isoforms 2-5, 12-15 kDa (predicted)
• Mouse (Q60765): 20 kDa (predicted)
• Rat (P29596): 20 kDa (predicted)
• Existence of phosphorylation and other modification sites.

WB experiment tips

Precautions

• ATF3 signal is easily observed in some cell lines, such as 293T; in some cell lines, the signal is weak or absent, but the signal is significantly enhanced after stimulation induction, such as Raw264.7, THP-1; ATF3 is difficult to detect in some normal tissues, such as liver and heart tissues, but literature reports that the expression of ATF3 in tissues is enhanced after stimulation. Please note that there may be differences in the optimal induction methods for different cells or tissues. Please choose the appropriate stimulation induction conditions according to the literature or instructions.
• ATF3 is located in the nucleus. We recommend using ultrasonic disruption to obtain more target proteins from cells. If the signal is still weak in the whole cell lysate, we recommend trying to extract the nuclear fraction to enhance the detection signal.
• The actual molecular weight of ATF3 is 21 kDa. Please follow the experimental suggestions for small molecular weight proteins for operation. See the key control points below for details.

Positive control

• HEK-293T whole cell lysate
• 80nM TPA induction overnight, followed by 1 µg/mL LPS for 8 hours
• RAW264.7 whole cell lysate treated with 1 µg/mL LPS for 2 hours

Example of results

Figure 3: Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434).

Lane 1: 20 µg of 293T whole cell lysate.
Lane 2: 20 µg of human liver tissue lysate.
Lane 3: 20 µg of Raw264.7 whole cell lysate.
Lane 4: 20 µg of mouse liver tissue lysate.
Lane 5: 20 µg of MEF whole cell lysate.
Lane 6: 20 µg of mouse heart tissue lysate.

Predicted band size: 21 kDa.
Detected band size: 21 kDa.

Figure 4: Anti-ATF3 antibody [EPR19488] - ChIP Grade (ab207434).

Lane 1: 20 µg of unstimulated THP-1 whole cell lysate.
Lane 2: 20 µg of THP-1 whole cell lysate induced with 80nM TPA overnight, followed by treatment with 1ug/ml LPS for 8 hours.
Lane 3: 20 µg of unstimulated Raw264.7 whole cell lysate.
Lane 4: 20 µg of RAW264.7 whole cell lysate treated with 1 µg/mL LPS for 2 hours.

Detected band size: 21 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. Ultrasonic treatment of cells to enrich the target protein.
3. Keep the sample on ice throughout the sample preparation process.
4. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.
5. We strongly recommend using a positive control.

Electrophoresis:

1. For target proteins with smaller molecular weights (such as molecular weight <25 kDa), use a 15% separation gel for electrophoresis.
2. Load at least 20 μg total protein for electrophoresis.

Transfer:

1. For target proteins with smaller molecular weights, it is recommended to use a 0.22 μm PVDF membrane.
2. We recommend using Coomassie Brilliant Blue staining after transfer to confirm the success of the transfer (if fluorescence labeling detection is chosen, make sure Coomassie Brilliant Blue is completely washed off).

Antibody incubation:

1. Please choose the optimal antibody working concentration according to the product manual.

References

1. B P Chen, G Liang, J Whelan, T Hai. ATF3 and ATF3 delta Zip. Transcriptional repression versus activation by alternatively spliced isoforms. Comparative Study J Biol Chem. 1994 Jun 3;269(22):15819-26.
2. Yoshinori Hashimoto, etc. An alternatively spliced isoform of transcriptional repressor ATF3 and its induction by stress stimuli. Nucleic Acids Res. 2002 Jun 1;30(11):2398-406. doi: 10.1093/nar/30.11.2398.
3. B P Chen, C D Wolfgang, T Hai. Analysis of ATF3, a transcription factor induced by physiological stresses and modulated by gadd153/Chop10. Mol Cell Biol. 1996 Mar;16(3):1157-68. doi: 10.1128/MCB.16.3.1157.
4. Heng Zhou, etc. Activating transcription factor 3 deficiency promotes cardiac hypertrophy, dysfunction, and fibrosis induced by pressure overload. PLoS One. 2011;6(10):e26744. doi: 10.1371/journal.pone.0026744. Epub 2011 Oct 28.
5. Ryosuke Takii, etc. Heat shock transcription factor 1 inhibits expression of IL-6 through activating transcription factor 3. J Immunol. 2010 Jan 15;184(2):1041-8. doi: 10.4049/jimmunol.0902579. Epub 2009 Dec 16.
6. Mohamed S Hasim, etc. Activating Transcription Factor 3 as a Novel Regulator of Chemotherapy Response in Breast Cancer. Transl Oncol. 2018 Aug;11(4):988-998. doi: 10.1016/j.tranon.2018.06.001. Epub 2018 Jun 22.