A, schematic representation of Ac45-FLAG, Ac45ΔC-FLAG,
a3-cMyc, EYFP-c, and EYFP-c″ constructs used for immunoprecipitation
assays. B, immunoprecipitation analysis showing that Ac45 and
Ac45ΔC interact with a3, c, and c″, respectively. C,
schematic representation of Ac45-EYFP, Ac45ΔC-EYFP, Rluc-a3, Rluc-c,
Rluc-c″, and Rluc-d1 constructs used for BRET assays. D,
Western blot analysis of COS-7 cells transfected with EYFP, Ac45-EYFP, or
Ac45ΔC. Transfected COS-7 cells were lysed in standard sample buffer,
and cell lysates (30 μl) were subjected to analysis by SDS-PAGE and
immunoblotting with anti-GFP rabbit polyclonal antibody. E, BRET
assays showing the levels of interaction of Ac45 with V0 subunits
a, c, c″, and d1. COS-7 cells co-expressing Ac45-EYFP or
Ac45ΔC-EYFP and either Rluc tagged a3, c, c″, or d1 subunits were
assayed following the addition of coelenterazine and the BRET ratio relative
to the Rluc alone expressing cells determined (normalized BRET ratio).
Similarly, the normalized BRET ratio was determined for cells co-expressing
EYFP with each of the Rluc-tagged subunits. The data represent the means from
six independent experiments ± S.E. p values < 0.05 indicate
significant differences between Ac45-EYFP and EYFP alone control. p
values < 0.05 indicate the significant differences between Ac45 mutant and
wild type (WT) Ac45.