Fig. 2

FBXO9 is closely associated with V-ATPase. A Workflow diagram representing the FBXO9 interactor search process using MS. B List of top proteins identified as potential binders to FBXO9. C Co-IP of different subunits (HA-tagged) of the V-ATPase V1 domain. The V-ATPase V1 domain subunits were immunoprecipitated using anti-HA beads, and the presence of bound FLAG-Fbxo9 was detected using anti-FLAG antibodies. D Co-IP of FLAG-Fbxo9 with HA-Atp6v1a in HEK293T cells. FLAG-Fbxo9 was immunoprecipitated using anti-FLAG antibodies, and the presence of bound HA-Atp6v1a was detected using anti-HA antibodies. E Schematic diagram illustrating the design of full-length HA-tagged ATP6V1A (FL) and a series of truncated, overlapping constructs (left). HEK293T cells were co-transfected with these constructs, followed by IP assay using anti-HA beads. Immunoblots were then probed with both anti-FLAG and anti-HA antibodies to detect the expression of the constructs (right). F Schematic diagram demonstrating the design of full-length FLAG-tagged FBXO9 (FL) and multiple truncated, overlapping constructs. HEK293T cells were co-transfected with these constructs and subsequently immunoprecipitated with anti-FLAG beads. Immunoblotting was conducted using anti-HA and anti-FLAG antibodies to detect the expression of the constructs (right). G Co-IP of the endogenous ATP6V1A with ectopically expressed FLAG-FBXO9 was conducted in HEK293T cells