Ca_v1.2 α_1c interacts and colocalizes with SARS-CoV-2 S protein and ACE2.

(A) HEK293 cells were co-transfected with Ca_v1.2 α_1c-Flag and SARS-CoV-2 S-Myc, and then subjected to immunoprecipitation (IP) by using anti-Flag agarose beads. Representative western blots of whole-cell lysates and eluates after IP are shown. (B) The co-immunoprecipitation of Ca_v1.2 α_1c-Flag and the SARS-CoV-2 S protein truncation mutant SARS-CoV-2 S1-Myc. (C) The co-immunoprecipitation of Ca_v1.2 α_1c-Flag and ACE2-Flag with the SARS-CoV-2 S protein truncation mutant SARS-CoV-2 RBD-Myc. (D) Purified soluble His-tagged SARS-CoV-2 S1 protein was pooled with the lysate from Ca_v1.2 α_1c-Flag transfected HEK293 cells and then pulled-down by using anti-Flag agarose beads. (E) The co-immunoprecipitation of Ca_v1.2 α_1c-Flag and ACE2-Myc. (F and G) Immunofluorescence assay. Vero-E6 cells were transfected with Ca_v1.2 α_1c-Flag for 24 h, then cells were incubated with SARS-CoV-2 (M.O.I. = 10) at 4°C for 1 h. The images comprising three single fluorescence channels were analysis by using Imaris software. The arrowhead indicates the representative colocalization of Ca_v1.2 α_1c-Flag (green), ACE2 (purple), and SARS-CoV-2 nucleocapsid protein (red) (F). The colocalization of Ca_v1.2 α_1c-Flag, ACE2, and SARS-CoV-2 nucleocapsid protein (indicated by the yellow arrowhead) is shown in three dimensions (G). The data shown are representative of three independent experiments.