Fig. 2.

Direct interaction of full-length ARNO with aldolase and all 4 a-subunit isoforms of vacuolar-type H⁺-ATPase (V-ATPase). A and B: direct interaction of ARNO with aldolase was further confirmed in pull-down (PD) experiments with recombinant proteins. First (A), ALDO-B was in vitro translated and metabolically labeled with fluorescent BODIPY (6His-ALDO-BODIPY; fourth lane). In this pull-down experiment, the ALDO-B was detected by laser scanning fluorescence analysis (FL) in the first lane (GST-ARNO) but not in the second and third lanes (GST-only and beads as controls). Second (B), ALDO-B was in vitro translated without metabolic labeling (6His-ALDO). In this pull-down experiment, the ALDO-B was detected only in the first lane (GST-ARNO), but not in the control lanes (GST-only and beads as controls), by Western blot analysis (WB) using an anti-ALDO-A/B (D-18) antibody. C: direct interaction of ARNO with all 4 a-subunit isoforms demonstrated in pull-down experiments using recombinant proteins. Cytosolic tails of a1-, a2-, a3-, and a4-subunit isoforms (a1N, a2N, a3N, and a4N, respectively) were in vitro translated and metabolically labeled with [³⁵S]methionine and used in pull-down assays with GST-ARNO(wt). Detection of a1N, a2N, a3N, and a4N was performed by autoradiography (AR). Representative data of 3 independent experiments are shown.