Apc 039

mCCDcl1 cells were seeded on to Corning Costar^™ Transwell^™ Permeable Supports (0.4 μm pore size) in 24 well plates at 1×10⁵ cells/cm² in complete growth medium. After 7–9 days, cell monolayers were washed in PBS for two times and fixed in 100% methanol (chilled at -20°C) for 5 min at RT, followed by three times wash in ice cold PBS for 5 min each as done before [27 (link), 34 (link)]. The cells were then blocked in blocking buffer (PBS, 10% goat serum and 0.05% Triton X-100) for one hour. Whereupon primary antibodies were add to the blocking buffer at 1:100 dilution and incubated overnight at 4°C. The following primary antibodies were used: anti-SK1 (Alomone, APC-039), anti-SK3-ATTO-594 (Alomone, APC-025-AR), anti-IK1 (Alomone, ALM-051), anti-BKα (Alomone, APC-021) and anti-TRPV4 (Alomone, ACC-034). After primary antibody incubation, the cells were washed 3 times in PBS, 5 min each, followed by incubation with secondary antibody (Life Technologies, alexa fluor 488 or 594 labeled goat anti-rabbit) in blocking buffer. The cells were then washed another three times in PBS, 5 min each, and mounted with ProLong Gold Antifade Mountant with DAPI (Life Technologies). Cells were imaged at 100x using a Nikon A1R Confocal Laser Microscope or a Zeiss Axioskop 40 microscope equiped with a AxioCam MRm CCD camera.

Using immunblot methods as previously described [21 (link), 34 (link)], two times Laemmli Sample Buffer (Bio-Rad) with 5% fresh β-mercaptoethanol was mixed with 1 part of cell pellet, and then heated for 10 min at 95°C with votexing every 3 min during the heating process. The sample was centrifuged at 12,000 rpm for 2 min. The supernatant was laded to 4–15% SDS-PAGE gradient gel, transferred to PVDF membrane, and blocked with 3% nonfat milk for 1 hour at RT. Membranes were incubated with anti-TRPV4 (Alomone, ACC-034), anti-SK1 (Alomone, APC-039), anti-SK3 (Alomone APC-025), anti-IK1 (ThermoFisher PA5-33875) and anti-BKα (Alomone APC-021) antibody at 1:1000, respectively, at 4°C overnight. After washing, membranes were incubated with goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) 1: 2000 for 1 h. All studies were performed in duplicate or triplicate.