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

Abstract

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.