Fig. 3

FBXO9 promotes non-degradative ubiquitination of ATP6V1A. A-C Ubiquitination assays in HEK293T cells were performed under different conditions. Co-transfection with HA-Atp6v1a (A) or HA-ATP6V1A (B) and His-Ub, along with Flag-FBXO9 after knockdown of FBXO9 using independent shRNAs (shFBXO9-2# and-3#) (C). Samples recovered with Ni-NTA (top) and whole cell lysate samples (bottom) were subjected to blotting against anti-HA or Flag as indicated. D Involvement of SKP1 and Cullin-1 in FBXO9-mediated ATP6V1A ubiquitination. HEK293T cells were transfected with His-Ub plasmids and shRNA targeting SKP1 or Cullin-1. Cell lysates were subjected to Ni-NTA bead pulldown, and the precipitates were analyzed by immunoblotting. E-G Effect of FBXO9 overexpression on exogenous (E, F) and endogenous (G) ATP6V1A protein levels. Cells were co-transfected with specific plasmids, and immunoblotting was carried out 48 h after transfection. H, I Effect of FBXO9 knockdown on ATP6V1A protein levels. A549 cells (H) and 889DTC cells (I) were depleted of FBXO9 or Fbxo9 using shRNA, and immunoblotting was performed. J, K Impact of FBXO9 expression on ATP6V1A half-life. HEK293T cells were transfected with FLAG-FBXO9 and subsequently treated with CHX for specific time periods, followed by immunoblotting analysis (J). The density of the ATP6V1A band was quantified to determine the ATP6V1A half-life (K). L Effect of neddylation inhibition on the ATP6V1A protein level in HEK293T cells. Cells were treated with MLN4924 for 12 h to inactivate the CRL ubiquitin ligase. Cell lysates were then analyzed by immunoblotting. M–O Ubiquitination of HA-ATP6V1A1 was measured in cells co-expressing FLAG-FBXO9 and different forms of His-Ub, as described in (A-C).