Panel A shows immunoblot images of GST pull-down assays using GST or GST-Arl8b loaded with GTP or GDP, detecting interaction with TBC1D9B from HEK293T lysates. Panel B shows immunoblot images of endogenous immunoprecipitation comparing TBC1D9B interaction with Arl8b in wild-type and Arl8b knockout HeLa cells. Panel C shows schematic diagrams of TBC1D9B domain architecture and deletion constructs highlighting GRAM domains, TBC domain, and EF-hand region. Panel D shows immunoblot images of GST pull-down assays using GST-Arl8b (Q75L) with GFP-tagged TBC1D9B full-length and truncation mutants. Panel E shows immunoblot images of co-immunoprecipitation assays assessing interaction between Arl8b (Q75L) and TBC1D9B wild-type or mutant proteins in HEK293T cells. Panel F shows fluorescence microscopy images of HeLa cells co-expressing GFP or GFP-TBC1D9B constructs with Arl8b (Q75L )or T34N MitoID, highlighting subcellular localization and recruitment patterns.
TBC1D9B interacts with Arl8b via its N-terminal region encompassing the GRAM1 domain. (A) GST and GST-Arl8b proteins were immobilized on glutathione resins and loaded with GTP or GDP, followed by incubation with HEK293T cell lysates. The precipitates were IB with anti-TBC1D9B antibody, and Ponceau S staining was done to visualize purified proteins. (B) WT and Arl8b KO HeLa cell lysates were subjected to endogenous IP using anti-Arl8b antibody, followed by IB with the indicated antibodies. (C) Domain architecture of TBC1D9B and its domain deletion mutants showing N-terminal GRAM1 and GRAM2 domains, TBC domain, and C-terminal EF hand. The image was created using IBS 2.0: Illustrator for Biological Sequences. (D) GST and GST-Arl8b (Q75L) proteins were immobilized on glutathione resins, followed by incubation with HEK293T cell lysates expressing GFP-tagged TBC1D9B or its domain deletion mutants. The precipitates were IB with anti-GFP antibody, and Ponceau S staining was done to visualize purified proteins. (E) HEK293T cell lysates expressing the indicated proteins were immunoprecipitated with anti-HA antibody and IB with the indicated antibodies. The values represent densitometric analysis of indicated proteins normalized to input and direct IP of Arl8b (Q75L)-HA. (F) HeLa cells were co-transfected with GFP alone or GFP-TBC1D9B (WT, 1–270 aa, or E91A) with Arl8b (Q75L or T34N)-MitoID (HA-tagged) and immunostained with an anti-HA (magenta) antibody. Scale bar: 10 µm. Source data are available for this figure: SourceData F5.