Figure 3.

Co-immunoprecipitation of VHA-E and VHA-A with VHA-a. Tonoplast enriched membranes of M. crystallinum were solubilized in buffer supplemented with 2% (v/v) Triton X-100. Antibody directed against VHA-a, either the N-terminal or membrane part, was added and immunoprecipitation was performed. The pellet samples and part of the supernatant (10–15% of total) were loaded on a SDS-gel, and Western blot was performed with anti VHA-E or A. The band intensities related to loaded sample size indicate that only a fraction of total V-ATPase was immuno-precipitated with the anti-VHA-a antibodies. As controls, immunoprecipitation was performed without added serum and with preimmune serum. With anti-E, monomeric and dimeric band of 30 and 60 kDa was detected by Western blotting in the precipitate obtained with anti VHA-a_N-term, with anti VHA-A, a 65 kDa band was labelled in separations of both, the precipitates obtained with anti VHA-a_N-term and anti-VHA-a_Memb, respectively.