Recombinant PEPD Protein Expression and Characterization

hPEPD, hPEPD-G278D, hPEPD-R265X and hPEPD-X265R were produced in E.coli as previously described [13 (link), 17 (link)]. EP and human EGF (236-EG-200) were purchased from Fresenius Kabi and R&D Systems, respectively. The following antibodies were used in the study: anti-PEPD (Abcam, ab86507) which detects hPEPD, hPEPD-G278D and mPEPD, anti-ErbB1 (Cell Signaling, 2232), anti-p-ErbB1 (Y1173) (Cell Signaling, 4407), anti-ErbB2 (Cell Signaling, 2165), anti-p-ErbB2 (Y1221/1222) (Cell Signaling, 2243), anti-AKT (Cell Signaling, 4691), anti-p-AKT (Cell Signaling, 4060), anti-ERK (Cell Signaling, 9102), anti-p-ERK (Cell Signaling, 9101), anti-STAT3 (Cell Signaling, 4904), anti-p-STAT3 (Cell Signaling, 9145), anti-cleaved caspase-3 (Cell Signaling, 9661), anti-BCL-2 (Cell Signaling, 2870), anti-BAX (Cell Signaling, 2772), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Millipore, MAB374), and biotin-conjugated anti-6XHistidines (His)-tag (Bethyl, A190-113B). Horseradish peroxidase (HRP)-conjugated streptavidin (N100) was purchased from Thermo Scientific. A goat anti-rabbit IgG-HRP was purchased from Jackson ImmunoResearch (111-035-003).