Polyjet

HEK 293 cells were originally obtained from ATCC and cultured and transfected essentially as previously described (15 (link), 16 (link), 17 (link)). The cells were maintained in DMEM containing 10% fetal bovine serum (both Life Technologies), incubated at 37 °C in 5% CO₂, and used between passage 18 and 30. HEK293 cells used in this study do not express endogenous BK channel subunits as determined by mRNA, protein, or functional assays (15 (link), 16 (link), 17 (link)). For transfection, the cells were typically plated on 6-well plates for 24 h before transfection with corresponding plasmid cDNA using PolyJet (tebu-bio). In all co-transfection assays, channel cDNAs were transfected at a 1:1 ratio with acyl thioesterases or empty pcDNA3.1 plasmid in controls.

HEK 293 cells were cultured and transfected as previously described (12 (link), 34 (link)). The cells were used between passage 18 and 30 and originally obtained from ATCC. HEK293 cells used in this study do not express endogenous the BK α- or β1-subunit as determined by mRNA, protein, or functional assays (11 (link), 12 (link), 19 (link), 34 (link)).
The cells were plated on 96-well plates for On-Cell Western assays or on 12-mm coverslips in 6-well plates for electrophysiological experiments, maintained in DMEM containing 10% fetal bovine serum (both Life Technologies), and incubated at 37 °C in 5% CO₂. The cells were transfected 24 h after plating with the BK channel α-subunit or co-expressed with β1-subunit in a 2:1 ratio of cDNA using Lipofectamine 2000 (Thermo Fisher) or Polyjet (tebu-bio) unless otherwise specified. All expression constructs were previously described (12 (link), 34 (link)). The cells were used for experiments 24–48 h post-transfection.

HEK293T cells cultured in Dulbecco modified Eagle medium (Fisher) with 10% fetal bovine serum (Fisher) were transfected with the Reelin or R3-6 cDNA constructs (Jossin et al., 2004 (link)), using Polyjet (Tebu-Bio). After 24 hr, the medium was replaced with a serum-free medium, which was collected 2 days later and stored at 4°C in the presence of a protease inhibitor cocktail (Complete, Roche). Prior to use, the supernatants were concentrated using Amicon Ultra columns with 100,000-molecular weight cutoff filters (Millipore) to reach the approximate concentration of 400 pM, which was estimated as described previously (Jossin et al., 2004 (link)), and dialyzed against culture medium by drop dialysis (Millipore VSWP02500). Mock solutions were prepared from control transfected HEK293T cells and used to control for potential co-purifying proteins.