Figure 1: CD36 protein structure.

CD36 Target Introduction

Protein Function

• CD36 is a transmembrane glycoprotein with multiple post-translational modification sites; it can bind to different ligands, including apoptotic cells, thrombospondin-1 (TSP-1), and fatty acids (FAs).
• CD36 is a scavenger receptor that can absorb long-chain fatty acids (LCFAs) and oxidized low-density lipoprotein (ox-LDL), and is involved in regulating lipid uptake, immune recognition, inflammation, molecular adhesion, and apoptosis.
• CD36 is expressed in various cell types, such as platelets, monocyte phagocytes, adipocytes, hepatocytes, myocytes, and some epithelial cells.
• CD36 promotes tumor metastasis and accelerates tumor growth. In tumor tissues, CD36 is expressed in tumor cells, MVECs, stromal cells, and immune cells, and the expression levels of CD36 vary among different cell types.

Protein characteristics

• CD36 can undergo various post-translational modifications, such as glycosylation, phosphorylation, palmitoylation, acetylation, and ubiquitination, which regulate the stability, protein folding, and translocation of CD36.
• CD36 has ten potential glycosylation sites. Fully glycosylated CD36 is a transmembrane glycoprotein receptor with a molecular weight of approximately 88 kDa.

Protein localization

• Cell membrane
• Intracellular vesicles, endoplasmic reticulum, and mitochondria
• Golgi apparatus

Figure 2: IHC experimental results of CD36, Anti-CD36 primary antibody (ab133625). Human myocardial tissue sections (4% PFA-fixed paraffin-embedded tissue sections). Heat-induced antigen retrieval was performed (Tris/EDTA buffer, pH 9.0). Primary antibody was diluted at a ratio of 1/10,000 (0.17µg/ml). The positive staining signal is mainly observed in human myocardial endothelial cells.

Isoforms and post-translational modifications

• Human (P16671): Isoforms 1,3,4: 46-53 kDa (predicted), Isoform 2: 32 kDa (predicted)
• Mouse (Q08857): 52.7 kDa (predicted)
• Rat (Q07969): 52.7 kDa (predicted)
• N-glycosylation and O-glycosylation, with a ratio of approximately 2:1.
• Ubiquitinated at Lys-469 and Lys-472 sites.
• Phosphorylated, palmitoylated, and acetylated.

WB experiment tips

Precautions

• The CD36 protein has multiple sites that can be post-translationally modified, so there may be multiple bands
• Due to the various post-translational modifications of CD36, the size of the detected bands may not match the predicted size.
• CD36 undergoes ubiquitination at Lys469 and Lys472, including both polyubiquitination and monoubiquitination. In general, protein polyubiquitination leads to protein degradation by directing the target protein to the proteasome. CD36 can also be degraded through the autophagy-lysosome pathway. This can lead to the appearance of multiple bands
• The immunogen used for some CD36 products may have high homology with CD36/SCARB1 and CD36/SCARB2. However, the cross-reactivity of antibody products with these proteins may not have been experimentally confirmed. Therefore, there may be multiple bands in the WB experiment, and it is recommended to set positive and negative controls.
• The expression level of CD36 in normal brain tissue may be relatively low.

Positive control

• HepG2, 3T3-L1, and NIH 3T3 whole cell lysates
• Rat spleen and adipose tissue lysates

Negative control (no signal or weak signal)

• Rat brain tissue lysate
• Ramos and Jurkat whole cell lysates

Example of results

Figure 3: WB- Anti-CD36 antibody [EPR22512-58] (ab252923).

Primary antibody: Anti-CD36 primary antibody, diluted 1/1000.
Blocking: Blocked with 5% NFDM/TBST blocking solution.

Lane 1: Human placental tissue lysate
Lane 2: Human lung tissue lysate
Lane 3: Human breast cancer tissue lysate.

Predicted band size: 53 kDa
Detected band size: 70-110 kDa

Figure 4: WB- Anti-CD36 antibody [EPR6573] (ab133625).

Primary antibody: Anti-CD36 primary antibody, diluted 1/1000.
Blocking: Blocked with 5% NFDM/TBST solution.

Lane 1: THP-1 whole cell lysate.
Lane 2: THP-1 cells treated with 100ng/ml PMA for 72 hours
Lane 3: Human adipose tissue lysate
Lane 4: HepG2 whole cell lysate
Lane 5: 3T3-L1 whole cell lysate
Lane 6: RAW 264.7 whole cell lysate
Lane 7: Mouse liver tissue lysate.

Predicted band size: 53 kDa
Detected band size: 78 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, you should bear the following key points in mind:

Sample preparation:

1. Add a sufficient amount of protease inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. For multi-pass transmembrane proteins, it is strongly recommended not to boil the sample.
4. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.
5. Set up positive and negative controls.

Electrophoresis:

1. Load at least 20μg total protein for electrophoresis.

Transfer:

1. We strongly recommend using Ponceau S staining after transfer to confirm the success of the transfer.

Reference

1. Silverstein RL, Febbraio M. CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. Sci Signal. 2009 May 26;2(72):re3. doi: 10.1126/scisignal.272re3.
2. Pepino MY, Kuda O, Samovski D, Abumrad NA. Structure-function of CD36 and importance of fatty acid signal transduction in fat metabolism. Annu Rev Nutr. 2014; 34:281-303. doi: 10.1146/annurev-nutr-071812-161220.
3. Choromańska B, Myśliwiec P, Choromańska K, Dadan J, Chabowski A. The role of CD36 receptor in the pathogenesis of atherosclerosis. Adv Clin Exp Med. 2017 Jul;26(4):717-722. doi: 10.17219/acem/62325.
4. Wang J, Li Y. CD36 tango in cancer: signaling pathways and functions. Theranostics. 2019 Jul 9;9(17):4893-4908. doi: 10.7150/thno.36037. eCollection 2019.
5. Thorne RF, Ralston KJ, de Bock CE, Mhaidat NM, Zhang XD, Boyd AW, Burns GF. Palmitoylation of CD36/FAT regulates the rate of its post-transcriptional processing in the endoplasmic reticulum. Biochim Biophys Acta. 2010 Nov;1803(11):1298-307. doi: 10.1016/j.bbamcr.2010.07.002.
6. Noushmehr H, D'Amico E, Farilla L, Hui HX, Wawrowsky KA, Mlynarski W, Doria A, Abumrad NA, Perfetti R. Fatty acid translocase (FAT/CD36) is localized on insulin-containing granules in human pancreatic beta-cells and mediates fatty acid effects on insulin secretion. Diabetes. 2005 Feb;54(2):472-81. doi: 10.2337/diabetes.54.2.472.
7. Tao N, Wagner SJ, Lublin DM.CD36 is palmitoylated on both N- and C-terminal cytoplasmic tails. J Biol Chem. 1996 Sep 13;271(37):22315-20. doi: 10.1074/jbc.271.37.22315.
8. Woo MK, Yang JW, Beltran C, Cho S. Cell Surface CD36 Protein in Monocyte/Macrophage Contributes to Phagocytosis during the Resolution Phase of Ischemic Stroke in Mice. J Biol Chem. 2016 Nov 4;291(45):23654-23661. doi: 10.1074/jbc.M116.750018.