Chemiluminescence luminal reagents

ST cells were lysed by lysis buffer and the protein concentration was determined with the BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific). Equivalent amounts of cell lysate protein (25 μg) were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to 0.22-μm nitrocellulose membranes (Pall, Port Washington, NY, United States). Then, the membranes were incubated with correspondent antibodies at 4°C overnight. After washing three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20, the membranes were incubated at room temperature for 2 h with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG. Detection was performed using chemiluminescence luminal reagents (Thermo Fisher Scientific).

PK-15 cells were lysed, and the protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equivalent amounts of cell lysate protein (30 μg) were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to 0.22-μm nitrocellulose membranes (Pall, Port Washington, NY, USA). Then, the membranes were incubated with mouse monoclonal or rabbit polyclonal antibodies at 37 °C for 1 h. After washing three times with phosphate-buffered saline (PBS) containing 0.05% Triton X-100, the membranes were incubated at 37 °C for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG. Detection was performed using chemiluminescence luminal reagents (Thermo Fisher Scientific).