*4.5. Protein Expression in PF-Treated Cells and Patient Tissues*

Total protein was measured using a modified Lowry method. Protein (7 µg for cells and 5 µg for tissues) was run on the automated Western blotting system, WES [78] (Cat No: 004-600, Protein Simple, San Jose, CA, USA). The primary anti-rabbit antibodies for EZH2 (1:50, Cat No: 5426S), FOXP3 (1:25, Cat No: 12632S), JARID2 (1:50, Cat No: 13594S), and H3K27me3 (1:25, Cat No: 9733S) (Cell Signaling, Danvers, MA, USA), anti-rabbit β-actin (1:100, Cat no: 4970S, Cell Signaling, Danvers, MA, USA), and anti-rabbit H3 (1:100, Cat No: 39451, Active Motif, Carlsbad, CA, USA) were used to measure expression levels within the samples. HRP-conjugated rabbit secondary antibody provided in the WES kit was used. Plates (12-230 kDa and 25 capillary) were run using default settings and results analyzed using the Compass for WES software (Version 5.0.1). Band area given by the software was used and normalized to β-actin or H3. Results were expressed as a ratio in which media alone (for cell treatments) or EuN (for patient tissues) was considered to be 1. It is important to note that proteins examined using the automated Western Blotting system, WES, will have different expected molecular weights compared to traditional Western blotting, due to differences in technology.
