Optimization of CTLA-4 and PD-1 proteins in EMT6 Mouse Mammary Cancer Cells by Western Blot


  • Nur Fatihah Ronny Sham Faculty of Medicine, Universiti Teknologi MARA, Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Narimah Abdul Hamid Hasani Faculty of Medicine, Universiti Teknologi MARA, Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Mohd Yusri Idorus Institute Medical Molecular Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA, Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Muhammad Khalis Abdul Karim Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
  • Syed Baharom Syed Ahmad Fuad Faculty of Medicine, Universiti Teknologi MARA, Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Harissa Husainy Hasbullah Faculty of Medicine, Universiti Teknologi MARA, Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
  • Mohammad Johari Ibahim Faculty of Medicine, Universiti Teknologi MARA, Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia




CTLA-4 and PD-1 proteins, EMT6 cells, Western blot


Targeting the activation of immune checkpoints is recognized as an effective strategy for triggering anti-tumour immune responses in cancer cells. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) were identified as potential crucial targets for cancer treatment. Overexpression of CTLA-4 and PD-1 proteins in primary tumour and human cell lines is well documented. In contrary, lack of data was available using animal cell lines. The presence study aims to optimize the expression of CTLA-4 and PD-1 proteins in EMT6 mouse mammary cancer cells using Western blot, and provide basic understanding of their association with breast cancer cell progression. Proteins extracted from EMT6 parental cells were adjusted to 30ng for gel electrophoresis. Afterwards, the protein was transferred to a nitrocellulose membrane for blotting. The membrane was then subjected to chemiluminescent for band detection. Results obtained using beta-actin as a housekeeping gene show that both CTLA-4 (32 kDa) and PD-1 (34 kDa) proteins were expressed by using a 1:1000 dilution for each antibody from the lysate of EMT6 mouse mammary cancer cells. The relative expression of PD-1 (4.0 ± 0.26) is higher compared to CTLA-4 (1.2 ± 1.8).  As a conclusion, both CTLA-4 and PD-1 proteins were indeed expressed in EMT6 mouse mammary cancer cells and this outcome provide the platform for extensive in vivo research on the link of both proteins with breast cancer using animal model.


P. Sharma and J. P. Allison. (2015). Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential. Cell, 161(2), 205-214. Doi: 10.1016/j.cell.2015.03.030.

A. Ribas and J. D. Wolchok. (2018). Cancer immunotherapy using checkpoint blockade. Science, 359(6382), 1350-1355. Doi: 10.1126/science.aar4060.

Y. Jiang, M. Chen, H. Nie, and Y. Yuan. (2019). PD-1 and PD-L1 in cancer immunotherapy: clinical implications and future considerations. Human Vaccines & Immunotherapeutics, 15(5), 1111–1122. Doi: 10.1080/21645515.2019.1571892.

K. Hudson, N. Cross, N. Jordan-Mahy, and R. Leyland. (2020). The extrinsic and intrinsic roles of PD-L1 and its receptor PD-1: Implications for immunotherapy treatment. Frontiers in Immunology, 11. Accessed: Dec. 28, 2022. [Online]. Available: https://www.frontiersin.org/articles/10.3389/fimmu.2020.568931.

M. E. Keir, M. J. Butte, G. J. Freeman, and A. H. Sharpe. (2008). PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol, 26, 677-704. Doi: 10.1146/annurev.immunol.26.021607.090331.

S. Laurent et al. (2013). The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-alpha production. Journal of Translational Medicine. Doi: 10.1186/1479-5876-11-108.

Y. Zheng, Y.-C. Fang, and J. Li. (2019). PD-L1 expression levels on tumor cells affect their immunosuppressive activity. Oncol Lett, 18(5), 5399-5407. Doi: 10.3892/ol.2019.10903.

I. Grenga, R. N. Donahue, L. Lepone, J. Bame, J. Schlom, and B. Farsaci. (2014). PD-L1 and MHC-I expression in 19 human tumor cell lines and modulation by interferon-gamma treatment. Journal for ImmunoTherapy of Cancer, 2(3), P102. Doi: 10.1186/2051-1426-2-S3-P102.

S. A. Rosenberg. 2014. IL-2: the first effective immunotherapy for human cancer. J Immunol, 192(12), 5451-5458. Doi: 10.4049/jimmunol.1490019.

A. Rotte. (2019). Combination of CTLA-4 and PD-1 blockers for treatment of cancer. Journal of Experimental & Clinical Cancer Research, 38(1), 255. Doi: 10.1186/s13046-019-1259-z.

P. Darvin, S. M. Toor, V. Sasidharan Nair, and E. Elkord. (2018). Immune checkpoint inhibitors: recent progress and potential biomarkers. Exp Mol Med, 50(12), 1-11. Doi: 10.1038/s12276-018-0191-1.

C.-K. Looi, F. F.-L. Chung, C.-O. Leong, S.-F. Wong, R. Rosli, and C.-W. Mai. 2019. Therapeutic challenges and current immunomodulatory strategies in targeting the immunosuppressive pancreatic tumor microenvironment. J Exp Clin Cancer Res, 38(1), 162. Doi: 10.1186/s13046-019-1153-8.

M. F. Sanmamed et al. (2015). Agonists of Co-stimulation in Cancer Immunotherapy Directed Against CD137, OX40, GITR, CD27, CD28, and ICOS. Semin Oncol, 42(4), 640-655. Doi: 10.1053/j.seminoncol.2015.05.014.

X. Wang, G. Guo, H. Guan, Y. Yu, J. Lu, and J. Yu. (2019). Challenges and potential of PD-1/PD-L1 checkpoint blockade immunotherapy for glioblastoma. J Exp Clin Cancer Res, 38(1), 87. Doi: 10.1186/s13046-019-1085-3.

B. Wang, L. Qin, M. Ren, and H. Sun. (2018). Effects of combination of anti-CTLA-4 and anti-PD-1 on gastric cancer cells proliferation, apoptosis and metastasis. Cell Physiol Biochem, 49(1), 260-270. Doi: 10.1159/000492876.

C. Brown et al. (2019). CTLA-4 Immunohistochemistry and quantitative image analysis for profiling of human cancers. J Histochem Cytochem, 67(12), 901-918. Doi: 10.1369/0022155419882292.

M. Sharma, Z. Yang, and H. Miyamoto. (2019). Immunohistochemistry of immune checkpoint markers PD-1 and PD-L1 in prostate cancer. Medicine (Baltimore), 98(38), e17257. Doi: 10.1097/MD.0000000000017257.

T. Mahmood and P.-C. Yang. (2012). Western blot: technique, theory, and trouble shooting. N Am J Med Sci, 4(9), 429-434. Doi: 10.4103/1947-2714.100998.

R. Kern and C. Panis. (2021). CTLA-4 expression and its clinical significance in breast cancer. Arch. Immunol. Ther. Exp., 69(1), 16. Doi: 10.1007/s00005-021-00618-5.

E. Contardi et al. (2005). CTLA-4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction. Int J Cancer. 117(4), 538-550. Doi: 10.1002/ijc.21155.

A. C. Urbano, C. Nascimento, M. Soares, J. Correia, and F. Ferreira. (2020). Clinical relevance of the serum CTLA-4 in cats with Mammary Carcinoma. Sci Rep, 10(1), Art. no. 1. Doi: 10.1038/s41598-020-60860-3.

C. G. Drake, E. Jaffee, and D. M. Pardoll. (2006). Mechanisms of immune evasion by tumors. Advances in Immunology, 90, 51-81. Doi: 10.1016/S0065-2776(06)90002-9.

X. Wu et al. (2019). Application of PD-1 blockade in cancer immunotherapy. Comput Struct Biotechnol J, 17, 661-674. Doi: 10.1016/j.csbj.2019.03.006.

S. Yang, Q. Zhang, S. Liu, A. R. Wang, and Z. You. (2016). PD-1, PD-L1 and PD-L2 expression in mouse prostate cancer. Am J Clin Exp Urol, 4(1), 1-8.

H. Gevensleben et al. (2016). PD-L1 promoter methylation is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients following radical prostatectomy. Oncotarget, 7(48), 79943-79955. Doi: 10.18632/oncotarget.13161.

Q. Liu, R. Cheng, X. Kong, Z. Wang, Y. Fang, and J. Wang. (2020). Molecular and clinical characterization of PD-1 in breast cancer using large-scale transcriptome data. Frontiers in Immunology, 11. Accessed: Dec. 28, 2022. [Online]. Available: https://www.frontiersin.org/articles/10.3389/fimmu.2020.558757.