Quantification of total and surface-associated hERG protein in cells maintained at lower temperatures

<p><b>Copyright information:</b></p><p>Taken from "Improved functional expression of recombinant human ether-a-go-go (hERG) Kchannels by cultivation at reduced temperature"</p><p>http://www.biomedcentral.com/1472-6750/7/93</p><p>BMC Biotechnology 2007;7():93-93.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241608.</p><p></p> Cells were cultured for 3 d and analyzed by flow cytometry. Data were averaged from 6 samples, each of 5000 cells (un-gated), and normalized against CHO hERG at 37°C. Normalized fluorescence intensity of fixed and permeablized CHO hERG (open bars) and untransfected control CHO cells (filled bars) at the respective temperatures stained with antibody C20 (which is raised against a C-terminal peptide sequence; A). Representative population fluorescence plots for CHO hERG cells from 37, 30 and 27°C in the same experiment as A (B). Normalized surface fluorescence intensity of non-fixed and non-permeablised CHO hERG (open bars) and control cells (filled bars) maintained at the respective temperatures stained with antibody 2110 (which is raised against a peptide between TM1 and TM2 which is predicted to be located on the surface of the plasma membrane; C)

Comparison of hERG activity measured by patch clamp and H-dofetilide binding in cells maintained at different growth temperatures

<p><b>Copyright information:</b></p><p>Taken from "Improved functional expression of recombinant human ether-a-go-go (hERG) Kchannels by cultivation at reduced temperature"</p><p>http://www.biomedcentral.com/1472-6750/7/93</p><p>BMC Biotechnology 2007;7():93-93.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241608.</p><p></p> CHO hERG cells grown at 37°C were split and kept subconfluent at 37, 30 or 27°C for 3 d (A,B). HEK293 hERG was split and kept at 37 or 30°C for 24 h (C). Mean tail currents recorded by patch clamping (IonWorksHT) from 4 independent experiments, 32–64 cells were patched for each data point in every experiment (A). H-dofetilide binding activity for CHO hERG and HEK hERG membrane preps respectively (B,C). Each data point was from 2 independent experiments, each of 3 repeats. Background (non-specific) binding was measured in the presence of an excess amount of non-radioactive dofetilide. Specific binding (open bars) was calculated as the total counts minus background counts and the total: background ratio (diamonds) was calculated as the total counts divided by the background counts

Ultrastructural characteristics of untransfected control and hERG transfected CHO cells

<p><b>Copyright information:</b></p><p>Taken from "Improved functional expression of recombinant human ether-a-go-go (hERG) Kchannels by cultivation at reduced temperature"</p><p>http://www.biomedcentral.com/1472-6750/7/93</p><p>BMC Biotechnology 2007;7():93-93.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241608.</p><p></p> CHO hERG or untransfected cells from 37°C were split and kept subconfluent for 3 d at 30°C or 27°C. At 30°C, hERG transfected CHO cells showed numerous large vesicle/vacuole-like structures (A,B), which were fewer in number in untransfected 30°C CHO cells (B). At 27°C, the number of vesicle/vacuole-like structures were reduced compared to the 30°C cells (C,D /A,B). Immunolocalized hERG + 5 and 10 nm colloidal gold secondary antibody was present on vesicle/vacuole-type membrane (E,F, respectively). hERG + 10 nm colloidal gold secondary labelling was present adjacent to golgi (F). cm, cell membrane; gl, golgi; nu, nucleus. Bar, A-D,5 μm. E,F, 250 nm. E inset, 100 nm