Goat anti rabbit

Kidney tissues (left whole kidneys) were homogenized in lysis buffer (320mM sucrose, 200mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1mM ethylenediaminetetraacetic acid, pH 7.2), protease inhibitor cocktail (Sigma-Aldrich Korea), and phosphatase inhibitor cocktail (Sigma-Aldrich Korea), using a Teflon homogenizer. Cellular debris was removed from homogenates by centrifuging at 3,000 rpm at 4°C for 10 minutes. Total protein contents were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (10μg) and transferred onto polyvinylidene difluoride membranes in a transfer buffer containing 250mM Tris-HCl, 4% SDS, 40% glycerol, 0.02% bromophenol blue, and 20% 1,4-dithiothreitol. Membranes were then blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 for 1 hour, incubated with specific primary antibodies for 24 hours, and then incubated with goat antirabbit (1:5,000, Thermo Fisher Scientific), rabbit antigoat (1:5,000, Invitrogen), or goat antimouse (1:10,000, Thermo Fisher Scientific) IgG HRP secondary antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific), and analyzed using Bio-1D software (SIM International Group, Newark, DE, USA).

Plates containing the PDAC cell lines were washed with PBS and lysed with Triton X-100 lysis buffer (25 mM Tris buffer, 100 mM NaCl, 1 mM EDTA, and 1% Triton X-100), supplemented with cOmplete protease inhibitor (Roche) and Phosphatase Inhibitor Cocktail (Sigma–Aldrich). Cells were incubated on ice for 10 min before scraping into a microcentrifuge tube and placed back on ice. Sample tubes were centrifuged for 10 min at 13,200 rpm at 4 °C. The supernatant was collected and used to determine protein concentration using a Bradford Assay (Bio-Rad), and samples were prepared with 4× Laemmli sample buffer (Bio-Rad). Samples were then boiled and stored at −20 °C. About 10% polyacrylamide gels were used to clarify lysates before transferring onto polyvinylidene fluoride membranes (Millipore; IPVH00010) at 90 V for 90 min. Membranes were blocked for 1 h in 5% bovine serum albumin solution diluted in TBST (TBS with 0.05% Tween-20) and washed with TBST. Membranes incubated overnight in primary antibodies diluted in 5% bovine serum albumin solution supplemented with sodium azide. Secondary antibodies used were goat antimouse (Invitrogen; 31432) and goat anti-rabbit (Invitrogen; 31462). Membranes were washed with TBST before imaging using the ChemiDoc MP Imaging System (Bio-Rad) and ECL reagent.