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.
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.
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