Sc 28259

Panc-02 and Panc-02-PTX cells were fixed with 4% paraformaldehyde after treatment or transfection at the indicated time points. Then we permeabilized the cells with 0.1% Triton100. After that cells were immersed three times in Tris-buffered saline (TBS) and incubated with the PHB1 primary antibody (sc-28259, diluted 1:200; Santa Cruz Biotechnology, TX) overnight at 4°C and the corresponding Cy3 goat anti-rabbit secondary antibodies (3101, diluted 1:150; Earthox, Millbrae, CA, USA) for 1 h at room temperature (RT). DAPI (diluted 1:1,000; Sigma-Aldrich) was used to stain the nucleus. Tissue sections were performed on a microtome (HM 430, MICROM International, Walldorf, Germany). Sections were blocked with 5% BSA in PBS for 30 min and incubated with the primary mouse monoclonal antibody anti-FoxM1 (sc-271746, diluted 1:100; Santa Cruz Biotechnology) or polyclonal rabbit antibody against PHB1 (sc-28259, diluted 1:200; Santa Cruz Biotechnology), respectively, at 4°C overnight. The primary antibodies were detected with the secondary antibodies (sheep anti-mouse or goat anti-rabbit). After immunolabeling, the tissue samples were incubated with 1 μg/mL DAPI and covered in 0.1% 1, 4-diazabicyclo [2.2.2] octane (DABCO; Sigma-Aldrich). Microscopic images of cells were obtained using an Olympus IX71.

Cultivated cells were harvested and lysed using lysis buffer [1% (v/v) Triton X-100, 200 mM HEPES (pH 7.9), 300 mM NaCl, 100 mM KCl, 10 mM EDTA, 10 µg/ml aprotinin, 100 µg/ml leupeptin, and 10 µM PMSF]. Twenty micrograms of total protein lysate was electrophoresed in an acrylamide gel and transferred to a TransBlot^® membrane (162–0145, Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% (w/v) milk, incubated with primary antibodies for 1 h at room temperature, washed and incubated for 1 h with the appropriate HRP-conjugated secondary antibodies (sc-2004 and sc2005, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The blots were washed in PBS containing 0.05% (v/v) Tween-20^® (P1379, Sigma-Aldrich Korea) and an ECL kit (34080, Pierce Biotechnology, Rockford, IL, USA) was used prior to detection of protein bands. The antibodies used were: rabbit anti-prohibitin (sc-28259, Santa Cruz Biotechnology, Inc.), rabbit anti-β-catenin (sc-7199, Santa Cruz Biotechnology, Inc.), rabbit anti-GSK-3β (sc-9166, Santa Cruz Biotechnology, Inc.), rabbit anti-p-GSK-3β (sc-11757-R, Santa Cruz Biotechnology, Inc.), mouse anti-c-Myc (sc-40, Santa Cruz Biotechnology, Inc.), and mouse anti-β-actin monoclonal (sc-8432, Santa Cruz Biotechnology, Inc.) to determine equal loading.