Fig. 1

Interaction of a subunit isoforms with Rab27A and A subunit. (A) Schematic illustration of the subunits of the mammalian V-ATPase. (B) Interaction of a subunit isoforms with Rab27A. FLAG-tagged a isoforms and various V5-fused forms of Rab27A were co-expressed in HEK293T cells. The cells were lysed, and lysates were immunoprecipitated with an anti-FLAG antibody. The precipitates were analyzed using antibodies against FLAG (upper panel) and V5 (middle panel). As a control, cells were co-transfected with an empty vector and a recombinant plasmid harboring V5-fused Rab27A (Control). WT, T23N, and Q78L indicate wild-type, GDP-bound form, and GTP-bound form Rab27A, respectively. In addition, 5% of the cell lysate was subjected to western blotting with an anti-V5 antibody (lower panel). Original blots are presented in Supplementary Fig. 7. The blots in (B) were cut horizontally at ~ 60 kDa. Upper and lower membranes were subjected to hybridization with anti-FLAG and anti-V5 antibodies, respectively. A full-length membrane was cut because the amount of protein in immunoprecipitation samples was insufficient to prepare multiple full-length membranes. (C) Interaction of the a2 subunit and Rab27A with the A subunit in V₁. FLAG-Rab27A(T23N) (lanes 1–3) and FLAG-a2 (lanes 4–6) were co-expressed with V5-a2 and V5-Rab27A(T23N), respectively. Immunoprecipitation was performed as described in (B). Original blots are presented in Supplementary Fig. 8. D Interaction of the a3 subunit and Rab27A with the A subunit in V₁. FLAG-Rab27A(T23N) (lanes 1–3) and FLAG-a3 (lanes 4–6) were co-expressed with V5-a3 and V5-Rab27A(T23N), respectively. Immunoprecipitation was performed as described in (B). Original blots are presented in Supplementary Fig. 8. (E) Presence of a2 and a3 isoforms and Rab27A in the membrane fraction. FLAG-Rab27A(T23N) and V5-a isoforms were co-expressed in HEK293T cells. The cells were lysed and centrifuged at 100,000 × g for 30 min to separate the membrane (M) and soluble (S) fractions. Immunoprecipitation was performed as described in (B). α-Tubulin was used as a control cytosolic protein. Original blots are presented in Supplementary Fig. 9.