Figure 2

Deletion of a non-conserved loop in the TLDc domain of Tbc1d24 and the C-terminal extensions of both Tbc1d24 and Tldc1, does not result in their interaction with V-ATPase in GST pull down assay. The GST-tagged full-length purified recombinant Tldc1 (2–455) does not interact with V-ATPase in GST pull-down assay. (A) Schematic representation of the domain architecture of Tbc1d24 and Tldc1 proteins and the constructs used to study the role of the non-conserved insertion in Tbc1d24 and the C-terminal extensions in both Tbc1d24 and Tldc1 in their interaction with V-ATPase. Boundaries of the domains, non-conserved regions and constructs are indicated as in Fig. 1. (B) Anti-B1 and anti-GST western blots of a representative GST pull-down assay, using the purified GST-tagged TLDc domains of Tbc1d24 (336–561) and Tldc1 (235–455), as well as, the GST-tagged truncated versions of the TLDc domains of Tbc1d24 and Tldc1: Tbc1d24 (336-446_496-556) and Tldc1 (235–410), and the GST-tagged full-length recombinant Tldc1 (2–455) with kidney lysate as the source of the B1 subunit of V-ATPase. GST only pull-down was used as a negative control; pull-down with the GST-tagged TLDc domain of Ncoa7 (775–943) was used as a positive control, anti-GST blot was used as a loading control for comparison between samples. This experiment was repeated three times with similar results. Longer exposure of anti-GST blot is shown to confirm the presence of a relatively low amount of GST-tagged Tbc1d24 (336–561) in the pull-down assay.