Fig. 2

F-ATPase/VGCC protein complex verification. a. 1D immunoblot analysis of the VGCC α2δ-1 subunit in neutrophils. The VGCC α2δ-1 subunit-containing protein complexes in a NativePAGE Novex 4–16% Bis-Tris gel were detected at approximately 750 kDa via immunoblotting, the same position as the F-ATPase F1 β-subunit. b. 2D immunoblot analysis of the F-ATPase and VGCC protein complex. Plasma membrane lysed by 2% digitonin was loaded and run on a BN-PAGE gel. One lane (Sample) was cut and equilibrated in SDS loading buffer, followed by fixation on a SDS-PAGE gel for 2D electrophoretic analysis. Two separate immunoblot analyses with a primary antibody against the VGCC α2δ-1 subunit or the F-ATPase F1 β-subunit were performed. The F-ATPase F1 β-subunit appears vertically below the VGCC α2δ-1 subunit in the same PVDF membrane. c. Immunofluorescence colocalization analysis of F-ATPase and VGCC. Representative images show that the VGCC α2δ-1 subunit and F-ATPase F1 β-subunit colocalized on the plasma membrane of neutrophils. d. Immunoprecipitation of VGCC α2δ-1 subunit and F-ATPase from plasma membrane protein in human neutrophils. Samples were immunoprecipitated with a mouse monoclonal antibody against α2 of the VGCC α2δ-1 subunit. F-ATPase was detected with a mouse monoclonal antibody against the F-ATPase F1 α-subunit or β-subunit and an HRP-conjugated goat anti-mouse IgG light chain specifically bound antibody. IgG control experiment did not detect F-ATPase