Fig 1: ITM2A interacts with v-ATPase. (A) Interaction between endogenous ITM2A and endogenous ATP6V0A4. HeLa cells were immunoprecipitated with either normal IgG or an anti-ITM2A antibody, followed by immunoblotting with an anti-ATP6V0A4 antibody (upper and middle panels) or anti-ITM2A antibody (lower panel). WCL, whole cell lysates. (B) Interaction between ITM2A and ATP6V0A4. HEK293 cells were transfected with HA-ITM2A, His-tagged ATP6V0A4 (His-ATP6V0A4) or both. ATP6V0A4 protein was immunopurified with an anti-His antibody and immunoprecipitates were immunoblotted with an anti-ITM2A antibody. (C) Interaction between ITM2A and endogenous ATP6V0A1. HEK293 cells were transfected with HA-ITM2A, and HA-ITM2A was immunopurified to examine the interaction with endogenous ATP6V0A1 (left). Simplified figure of v-ATPase complex is shown and ATP6V0A1, ATP6V0A4, and ATP6V1B1/ATP6V1B2 are indicated (right). (D) ITM2A is colocalized with ATP6V0A4. HEK293 cells were transfected with plasmid encoding Xpress-ATP6V0A4 in the presence or absence of plasmid encoding ITM2A. Cells were mock-treated, starved for 2 h or incubated with BafA1 (100 nM, 4 h) and immunostained with anti-Xpress (red) or anti-ITM2A (green) antibodies. Bars: 10 µm. (E) ITM2A is colocalized with ATP6V0A1. HEK293 cells were transfected with plasmid encoding Xpress-ATP6V0A1 in the presence or absence of plasmid encoding ITM2A. Bars: 10 µm. (F) Schematic diagram of ITM2A and its deletion mutants (N1 to N4, C1). Numbers correspond to the amino acid sequence. TM, transmembrane domain. (G) Identification of the region of ITM2A required for v-ATPase interaction. (i) HEK293 cells were transfected with Xpress-tagged ITM2A mutants (N1, N2, N3, N4), and Xpress-ITM2A mutants were immunopurified with anti-Xpress antibody. The immunoprecipitates were probed with anti- ATP6V0A1 antibody. (ii) HEK293 cells were transfected with Xpress-ITM2A wild type (WT) or Xpress-tagged ITM2A C1 (C1). Xpress-ITM2A proteins were immunopurified with an anti-Xpress antibody and immunoprecipitates were immunoblotted with an anti-ATP6V0A1 antibody.