Fig. 4. Truncation of the CNBD results in ER retention. (A) Schematic representation of HERG with six transmembrane segments (S1-S6). Downward arrow indicate residues of HERG corresponding to the putative CNBD, PAS domain (black arrows) and a previously identified segment involved in channel trafficking (gray arrows). HERG truncated constructs analysed in this study are indicated by the upward arrows. Maturation state corresponding to individual mutants is indicated as X (defective trafficking) or (normal trafficking). (B) Immunoblots of truncation constructs co-expressed with green fluorescent protein (GFP) to compare the transfection efficiency. Transiently transfected cells were lysed and equal amounts of solubilized protein were subjected to SDS-PAGE. The top membrane was probed with an anti-Myc antibody, stripped and reprobed for CASK to compare loading amounts (indicated as 15 µg and 30 µg). The bottom portion of the same membrane was probed for GFP. (C) Cells producing HERG_870X were lysed and subjected to overnight treatment with EndoH and PNGase. Treated (+) and untreated (–) cell lysates were separated on SDS-PAGE and subjected to immunoblotting with an anti-Myc antibody (-Myc). The arrow indicates the position of the mature band (M). (D) Cells transfected with the cDNAs encoding HERG_wt and HERG_750-860 were lysed 1 day after transfection and treated with EndoH (+) or untreated (–). Equal amounts of solubilized proteins were subjected to immunoblotting with an anti-HA antibody. (E) Cells producing different mutant constructs were co-transfected with GFP, lysed and subjected to immunoblotting. (top) The membrane was probed with anti-HA and anti-CASK antibodies. (bottom) The same membrane was probed with an anti-GFP antibody. The arrow indicates the position of CASK signal at the expected molecular weight.