Apc 055

Conventionally cultured Capan-1 cells were lysed in lysis buffer containing (mM) 250 TRIS, 1,25 NaCl, 20 NaF, 50 EDTA, 5% Trizol, and 1 × Sigma fast protease inhibitor (Sigma-Aldrich, Germany). The lysates were centrifuged for 15 min at 15000 g at 4 °C, and the supernatant was stored at − 80 °C until use. Protein concentration was determined in the samples by use of a Bradford assay. Ten to 20 μg of protein was loaded on 10% polyacrylamide precast gels (NuPAGE, Bis–Tris, Invitrogen), separated by electrophoresis, and transferred to a PVDF membrane (Invitrogen). Membranes were blocked in a solution with 3% BSA and 2% skim milk in TRIS-buffered saline containing 0.1% Tween (TBST) (60 to 90 min, room temperature (RT)). The membranes were incubated overnight at 4 °C with primary antibodies against KCNQ1 (1:1000 rabbit polyclonal, APC-022, Alomone Labs), TREK-2 (1:600 rabbit polyclonal, APC-055, Alomone Labs), and TASK-2 (1:2000 rabbit polyclonal, LS-C291082, (LifeSpan BioSciences, Inc.)), followed by rinsing in TBST and treatment with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000 to 1:2500 goat anti-rabbit, sc-2004 (Santa Cruz) in TBST with 3% BSA and 2% skim milk). Chemiluminescence of the immunoreactive bands was visualized with EZ-ECL (Biological Industries) on the Fusion FX (Vilber Lourmat) imaging system.

Confluent Capan-1 monolayers on membranes were fixed in 4% paraformaldehyde in PBS (phosphate-buffered saline) for 15 min at RT. The preparation was washed and treated with 0.1 M TRIS–glycine to reduce autofluorescence. Cells were permeabilized in PBS containing 0.2% Triton-X-100, rinsed once, and incubated with 5% BSA in PBS to block non-specific binding. Cells were then treated with primary antibodies against TREK-1 (1:50, sc-11557, Santa Cruz), TREK-2 (1:250, APC-055, Alomone Labs), and TASK-2 (1:75, APC-037, Alomone) overnight at 4 °C, followed by incubation with secondary Alexa488 conjugated antibodies (1:200) 1 h at RT. Negative controls were treated identically, except for the omission of primary antibody incubation. In the last steps, F-actin was labeled with Alexa Fluor 647 phalloidin (A22287, Molecular Probes) and nuclei stained with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, Molecular Probes). Membranes carrying the monolayers were cut out of the inserts and mounted on objective glasses with fluorescence mounting medium (S3023, DAKO). Fluorescence was investigated with a 63 × 1.2 NA objective on a Leica TCS SP5-MP confocal laser scanning microscope (Leica Microsystems, Heidelberg). Leica software was used to analyze and export images as TIFF files.