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Fig. 1. TMEM43 interacts with TASK-1 in the heterologous overexpression system. (A) Schematic diagram illustrating the heterologous DNA expression in HEK293T cell line and procedures of the co-IP assay. Tmem43, Cx26, and Cx30 coding sequences tagged with FLAG and Task-1 coding sequences were cloned into IRES2 vectors. Anti-TASK-1 antibody was used for immunoprecipitation, and the pulled-down protein was blotted with an anti-FLAG antibody. (B) Co-IP result, performed with normal IgG or TASK-1 antibody using lysates of HEK293T cells co-expressing TMEM43-FLAG and TASK-1. (C) Co-IP results of TASK-1 with Cx26-FLAG and Cx30-FLAG. Anti-FLAG antibody was used to detect the connexin channels. The first lane is a negative control. (D) A cartoon depicting the principle of the Duolink PLA. If the proteins of interest are in close proximity, the DNA probes hybridize to make circular DNA. This DNA can be amplified and visualized by fluorescently labeled complementary oligonucleotide probes. (E) Duolink PLA with anti-TMEM43 and anti-TASK-1 antibodies. Duolink PLA signal was recognized as far-red fluorescent (λex 644 nm, λem 669 nm), indicative of proximity of TMEM43 and TASK-1 (<40 nm). The lower panel is a negative control, using only an anti-TMEM43 antibody.
Exp Neurobiol 2021;30:319~328
© Exp Neurobiol