Figure 1: c-Fos protein structure.

c-Fos target introduction

Protein function

• c-Fos is a nuclear phosphoprotein that forms a heterodimer with c-Jun and then forms the AP-1 (activator protein-1) complex. This complex binds to specific AP-1 sites in the target gene promoter and enhancer regions of DNA, converting extracellular signals into changes in gene expression.
• c-Fos plays a key role in regulating cell development aimed at forming and maintaining the skeleton.
• c-Fos is believed to play an essential role in signal transduction, cell proliferation, and differentiation.

Protein characteristics

• Various extracellular stimuli such as growth factors, cytokines, neurotransmitters, peptide hormones, and stress can rapidly and transiently induce Fos protein expression.
• After stimulation, c-Fos is expressed immediately, but its lifespan is short, and the protein level disappears after a few hours.

Protein expression

• Expressed at very low levels in resting cells.
• When cells are stimulated to re-enter the growth phase, they undergo two waves of c-Fos expression. The first wave of protein expression peaks after 7.5 minutes of FBS induction and is localized to the endoplasmic reticulum. The second wave of protein expression occurs after 20 minutes of induction and peaks at around 1 hour, localized to the cell nucleus.

Protein localization

• c-Fos is expressed in the endoplasmic reticulum, cytoplasm, and cell nucleus.

Figure 2: ICC experimental results of c-Fos target, using Anti-c-Fos antibody [EPR21930-238] (ab222699). Green: c-Fos, Red: Tubulin, Blue: DAPI.

Isoforms & post-translation modifications

• Human (P01100): Isoforms 1-3: 29-41 kDa (predicted)
• Mouse (P01101): 41 kDa (predicted)
• Rat (P12841): 41 kDa (predicted)
• c-Fos undergoes phosphorylation and ubiquitination modifications.

WB experiment tips

Precautions

• The basal expression level of c-Fos is relatively low and needs to be stimulated for detection in WB experiments.
• Serum starvation, followed by treatment with 20% FBS for 2 hours or 200 nM PMA for 4 hours, can induce c-Fos expression in cells.
• The expression of c-Fos after stimulation is rapid and transient, so it is important to collect samples immediately after stimulation and prepare lysates to avoid protein loss.
• Multiple bands (50-65 kDa) may appear in stimulated or treated cell lysates, corresponding to different forms of c-Fos protein.

Positive control

• Serum starvation, followed by treatment with 20% FBS for 2 hours in HeLa cell lysate.
• Serum starvation, followed by treatment with 200 nM PMA for 4 hours in RAW264.7 cell lysate.
• Recombinant human c-Fos protein (ab56280).

Example results

Figure 3: WB- Anti-c-Fos antibody [2H2] (ab208942).

Primary antibody: used ab208942 at a concentration of 1/1000

Lane 1: Whole cell lysate of HeLa cells cultured overnight in serum starvation.
Lane 2: Whole cell lysate of HeLa cells cultured in serum starvation and then treated with 20% FBS for 2 hours.
Lane 3: Rat cortical neurons.
Lane 4: Rat cortical neurons treated with depolarizing buffer for 5 hours.

Predicted band size: 41 kDa.

Figure 4: WB- Anti-c-Fos antibody [EPR21930-238] (ab222699).

Primary antibody: used ab222699 at a concentration of 1/5000.

Lane 1: Whole cell lysate of RAW264.7 cells cultured overnight in serum starvation.
Lane 2: Whole cell lysate of RAW264.7 cells cultured overnight in serum starvation, then treated with 200 nM PMA for 4 hours.

Predicted band size: 41 kDa
Actual band size: 55-60 kDa

Key control points

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

Sample preparation:

• Add a sufficient amount of protease inhibitor to avoid degradation of the target protein.
• For phosphorylated c-Fos, add enough phosphatase inhibitor to prevent protein dephosphorylation during the extraction process.
• Use ultrasonic disruption to enrich c-Fos protein.
• Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Transmembrane:

• We strongly recommend using Coomassie Brilliant Blue staining after transmembrane to determine whether the transmembrane is successful.

IHC experiment tips

Precautions

• If using fluorescently labeled secondary antibodies for the experiment, we recommend using a blocking solution containing 1% BSA and a final concentration of 0.3 M glycine to quench the autofluorescence caused by aldehyde groups.
• Pay attention to the species of the primary antibody host and the species of the sample being tested. For example, when using a mouse host primary antibody ab208942 to detect mouse tissue, non-specific staining is prone to occur. In this case, you can choose to use a rabbit monoclonal antibody like ab222699 for the experiment. When performing mouse-on-mouse staining, appropriate IgG blocking reagents can also be added to prevent binding with endogenous IgG, thereby reducing non-specific staining.

Positive controls

• Rat hippocampal tissue
• Mouse cerebral cortex tissue

Example of results

Figure 5: IHC experimental results of c-Fos target, Anti-c-Fos antibody [EPR24046-20] (ab289723)

Sample name: Rat brain tissue paraffin section
Primary antibody: diluted 100 times (5.46 µg/mL)
Antigen retrieval method: Heat-induced antigen retrieval, Tris-EDTA buffer (pH 9.0)

Key control points

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

Sample fixation:

1. The sample fixation time depends on the size of the tissue block and the type of tissue, but for most samples, such as fixation with 4% PFA, fixing at room temperature for 18-24 hours is more appropriate.
2. Insufficient fixation can result in a higher signal at the edges of the sample and a weaker signal in the center, or even no signal.
3. Excessive fixation can block antigenic epitopes. Antigen retrieval can expose some of the epitopes. However, if the tissue fixation time is very long (more than a week), there may still be no signal after antigen retrieval.

Antigen retrieval:

1. We recommend using a pressure cooker for heat-induced antigen retrieval when performing immunohistochemistry experiments on paraffin sections. You can try fixing the sections at 110°C for 15 minutes. After retrieval, let it cool naturally and avoid putting it in cold water to prevent section detachment due to sudden temperature drop.
2. For tissues that are relatively brittle and prone to section detachment during high temperatures and high-pressure retrievals, such as bone and cartilage, or tissues with small cutting surfaces, such as the sciatic nerve, microwave retrieval can also be chosen.

Blocking:

1. If HRP conjugate is used for detection in subsequent steps, please treat the sections with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase.
2. Before incubating with the primary antibody, blocking with serum is required. Avoid choosing a blocking solution from the same species as the host. The source of the serum can be selected based on the host of the secondary antibody. For example, goat serum can be used as the blocking solution if the secondary antibody is Goat Anti-Rabbit IgG H&L (HRP polymer) or Goat Anti-Mouse IgG H&L (HRP polymer).

Antibody incubation:

1. If the experiment allows, perform a control experiment with only the secondary antibody without the primary antibody.
2. Incubate the fluorescently labeled secondary antibody in a dark box to avoid photobleaching.

References

1. Y Zhang, X H Feng, R Derynck. Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature. 1998 Aug 27;394(6696):909-13. doi: 10.1038/29814.
2. Guillaume Bossis, Cécile E Malnou, Rosa Farras, Elisabetta Andermarcher, Robert Hipskind, Manuel Rodriguez, Darja Schmidt, Stefan Muller, Isabelle Jariel-Encontre, Marc Piechaczyk. Down-regulation of c-Fos/c-Jun AP-1 dimer activity by Sumoylation. Mol Cell Biol. 2005 Aug;25(16):6964-79. doi: 10.1128/MCB.25.16.6964-6979.2005.
3. K Okazaki, N Sagata. The Mos/MAP kinase pathway stabilizes c-Fos by phosphorylation and augments its transforming activity in NIH 3T3 cells. EMBO J. 1995 Oct 16;14(20):5048-59.