Image 1: Structure of FOXP3

FOXP3 Target Introduction

Protein Function

• Transcriptional regulator, which is crucial for the development and inhibitory function of regulatory T-cells (Treg)
• It plays an essential role in maintaining homeostasis of the immune system by allowing the acquisition of full suppressive function and stability of the Treg lineage and by directly modulating the expansion and function of conventional T-cells.
• Depending on its interactions with other transcription factors, such as histone acetylases and deacetylases, it can act either as a transcriptional repressor or a transcriptional activator.
• Defects in FOXP3 cause immunodeficiency polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX).

Protein Characteristics

• Most tumor tissues are infiltrated with FOXP3-expressing Treg.

Protein Expression

• Down-regulated in regulatory T-cells (Treg) during inflammation.
• Highly expressed in the peripheral blood of cancer patients and most tumor tissues.

Protein Localization

• Predominantly expressed in the cytoplasm in activated conventional T-cells.
• Predominantly expressed in the nucleus in regulatory T-cells (Treg).

Figure 2: FOXP3 ICC experimental result image, Anti-FOXP3 antibody [2A11G9] (ab36607). Green: FOXP3, Red: actin filaments.

Isoforms & Post-translational modifications

• Human (Q9BZS1): Isoforms 1-4: 43-50kDa (predicted)
• Mouse (Q99JB6): 47kDa (predicted)
• Rat (D4Q8I2): 54kDa (predicted)
• Polyubiquitinated, leading to its proteasomal degradation in regulatory T-cells (Treg).
• Phosphorylation at Ser-418 regulates its transcriptional repressor activity and consequently, regulatory T-cells (Treg) suppressive function.
• Acetylation on lysine residues stabilizes FOXP3 and promotes differentiation of T-cells.

WB experiment tips

Precautions

• Since FOXP3 is only expressed in some tissues and cells, it is recommended to use a positive control for detection, such as HEK-293T cells transfected with wild-type FOXP3 vector.
• When using ab215206 for experiments, please note that this product:
• It is not suitable for detecting tissue samples derived from FOPX3.
• It is not suitable for detecting FOXP3 derived from rats.

Positive control

• HEK-293T transfected with FOXP3 (WT) expression vector with a GFP-myc tag, whole cell lysate.
• Recombinant Mouse Foxp3 protein.

Example of results

Figure 3: WB-Anti-FOXP3 antibody [EPR22102-37] (ab215206).

Lane 1: HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a GFP-myc tag, whole cell lysate
Lane 2: HEK-293T transfected with FOXP3 (WT) expression vector containing a GFP-myc tag, whole cell lysate.

Predicted band size: 47 kDa
Detected band size: 73 kDa

Key control points

In the experiment, along with addressing routine issues, it is essential to focus on the following key control points:

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Keep samples on ice throughout the entire sample preparation process.
3. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg of total protein from cell lysate or tissue homogenate.

Antibody incubation:

1. Select a suitable antibody working concentration according to the product datasheet.

IHC experiment tips

Precautions

• FOXP3 is mainly expressed in Treg cells, which are concentrated in the thymus, tonsils, and tumor tissues (such as breast, lung, liver, pancreatic, malignant melanoma, gastrointestinal, etc.). We recommend using human tonsil tissue as a positive control to confirm that the experimental system works appropriately.
• If ab215206is used for detection, we recommend using a polymer secondary antibody detection system, such as the Rabbit-specific IHC polymer detection kit HRP/DAB (ab209101), to obtain better detection results.

Positive control

• Human tonsil tissue

Example of results

Figure 4: IHC-Anti-FOXP3 antibody  [EPR22102-37] (ab215206)

Sample name: Paraffin-embedded rat thymus tissue
Antigen retrieval method: Heat-mediated antigen retrieval using ab208572 (Universal HIER antigen retrieval reagent).

Key control points

In the experiment, alongside routine issues, special emphasis should be placed on the following key control points:

Sample fixation:

1. The optimal fixation time for samples depends on the size and type of tissue, but for most samples, it is advisable to fix at room temperature for 18-24 hours.
2. Over-fixation can mask the epitope. While antigen retrieval can help overcome this masking, if the tissue is fixed for an extended period (eg, over a weekend), there may still be no signal even after antigen retrieval.

Antigen retrieval:

1. Antigen retrieval conditions should be optimized when performing immunohistochemistry experiments on paraffin sections. We recommend using a pressure cooker for heat-induced antigen retrieval. You can try to repair the sections at 110℃ for 15 minutes.

Reference

1. James B Wing, Atsushi Tanaka, Shimon Sakaguchi. Human FOXP3 + Regulatory T Cell Heterogeneity and Function in Autoimmunity and Cancer. Immunity. 2019 Feb 19;50(2):302-316. doi: 10.1016/j.immuni.2019.01.020.
2. Ling Lu, Joseph Barbi, Fan Pan.  The regulation of immune tolerance by FOXP3. Nat Rev Immunol. 2017 Nov;17(11):703-717. doi: 10.1038/nri.2017.75.
3. F Martin, S Ladoire, G Mignot, L Apetoh, F Ghiringhelli. Human FOXP3 and cancer. Oncogene. 2010 Jul 22;29(29):4121-9. doi: 10.1038/onc.2010.174. Epub 2010 May 24.
4. Hiroyoshi Nishikawa, Shimon Sakaguchi. Regulatory T cells in tumor immunity. Int J Cancer. 2010 Aug 15;127(4):759-67. doi: 10.1002/ijc.25429.
5. Oscar Palomares, Beate Rückert, Tuomas Jartti, Umut Can Kücüksezer, Tuomo Puhakka, Enrique Gomez, Heinz B Fahrner, Andreas Speiser, Andreas Jung, William W Kwok, Livije Kalogjera, Mübeccel Akdis, Cezmi A Akdis. Induction and maintenance of allergen-specific FOXP3+ Treg cells in human tonsils as potential first-line organs of oral tolerance. J Allergy Clin Immunol. 2012 Feb;129(2):510-20, 520.e1-9. doi: 10.1016/j.jaci.2011.09.031.
6. Estelle Bettelli, Maryam Dastrange, Mohamed Oukka. Foxp3 interacts with nuclear factor of activated T cells and NF-kappa B to repress cytokine gene expression and effector functions of T helper cells. Proc Natl Acad Sci U S A. 2005 Apr 5;102(14):5138-43. doi: 10.1073/pnas.0501675102.
7. Jorg van Loosdregt, Veerle Fleskens, Juan Fu, Arjan B Brenkman, Cornelis P J Bekker, Cornelieke E G M Pals, Jenny Meerding, Celia R Berkers, Joseph Barbi, Andrea Gröne, Alice J A M Sijts, Madelon M Maurice, Eric Kalkhoven, Berent J Prakken, Huib Ovaa, Fan Pan, Dietmar M W Zaiss, Paul J Coffer. Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity. Immunity. 2013 Aug 22;39(2):259-71. doi: 10.1016/j.immuni.2013.05.018.