Hamster anti muc1 ct ct2

NP-40 cell lysis buffer containing protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) was used to isolate proteins from pancreatic cancer cells. Equal concentrations of protein lysates (50 μg) were resolved using 4–20% denaturing polyacrylamide gels (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene fluoride (PVDF) membrane (Millipore EMD, Billerica, MA, USA) and blocked with 5% powdered milk in Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 (TBS-T). The following primary antibodies were used to probe the membrane for the protein expressions: rabbit anti-GalNAc-T5 (Abgent, San Diego, CA, USA, 1:1000), hamster anti-MUC1-CT (CT2, Abcam, USA, 1:1000), and mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA, 1:5000). After incubating with respective secondary antibodies, protein-antibody complex was detected using enhanced chemiluminescence (Bio-Rad, USA).

S2–013 MUC1F and S2–013MUC-CT cells were treated with variying concentration (1, 2.5 and 5μM) of p53 inhibitor Pifithrin-α hydrobromide (Tocris, MN, USA). After 24 hours of treatment, the cells were harvested and total proteins were isolated using RIPA buffer containing protease and phosphatase inhibitors. Wstern blotting analysis was performed as described above using the following primary antibodies: rabbit anti-GalNAc-T5 (Abgent, San Diego, CA, USA, 1:1000), hamster anti-MUC1-CT (CT2, Abcam, USA, 1:1000) and mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA, 1:5000).