Figure 2: Both KV10.1 demi-channels are detected in the same complex.
(a) Biotinylation of surface proteins and subsequent pull down of labelled molecules allowed the detection of full-length channel using an anti-KV10.1 C-terminal antibody, and also of the C-terminal truncated protein when expressed either alone or as split channel. (b) Co-immunoprecipitation of N- and C- terminal demi-channels. The N-terminal fragment was labelled with 5 × Myc and the C-terminal was 4 × HA tagged. Immunoprecipitation with HA-tag pulled down a fragment of size compatible with the N-terminal demi-channel (arrow), recognized by anti-Myc immunoblot. Asterisks indicate bands corresponding to the antibody used to immunoprecipitate. (c) Immunoprecipitation with anti-Myc also pulled HA-tagged fragments detected as a double band (arrows). The migration distance of the upper band was modified by deglycosylation (PNGase F lanes), as expected for the C-terminal fragment of KV10.1, which contains the glycosylated residues. Input lanes were loaded with the extract corresponding to half an oocyte; the equivalent to 30 oocytes were used to immunoprecipitate. (d) Native electrophoresis and immunoblot shows the presence of a complex with size similar to that of the continuous channel when demi-channels were expressed together, recognized by both anti-Myc and anti-KV10.1 antibodies.
