Fig. 2. AT3 interacts with hexameric assemblies of VCP.

a WB for VCP after BN-PAGE of whole cell lysates from HEK293T cells after transfection with control GFP, ASPSCR1, AT3, truncated ASPSCR1 (trASPSCR1, the portion included in AT3) or truncated TFE3 (trTFE3, the portion included in AT3). VCP hexamer size is ~582 kD (n = 3 biological repeats performed with similar result). b Mass spectrometry results after FLAG-IP from HEK293T cells transfected with tagged ASPSCR1 or AT3, showing VCP predominant in both, other than the peptides aligned with the FLAG-tagged bait itself, noted in red as ASPSCR1 or both ASPSCR1 and TFE3. c Negative stain TEM micrographs of FLAG-IP eluates after ASPSCR1 or AT3 overexpression in HEK293T cells (bars = 20 nm; biological repeats n = 3). d Reference-free 2D class averages of negatively stained particles recovered from FLAG-AT3 co-IP reveal intact VCP hexameric assemblies. Left and middle, VCP top or pore views (3620 and 2774 particles in each class, respectively); right, VCP side view (182 particles; box length and width = 25 nm). e Schematic of constructs of each type of AT3 as well as CΔ, a truncated portion of the carboxy half of ASPSCR1, not included in AT3 (HA human influenza hemagglutinin tag; NLS nuclear localization signal). Orientation from top-to-bottom is amino-to-carboxy termini. f VCP WB after BN-PAGE following transfection of HEK293T cells with ASPSCR1, CΔ, or an empty vector control as well as the last of these transfections treated subsequently with VCP inhibitor CB-5083 (biological repeats n = 3). g VCP WB after BN-PAGE following IP for TFE3-FLAG or AT3-FLAG with or without co-transfected CΔ, along with denaturing gel blots of input proteins below (biological repeats n = 3). h Negative stain TEM micrographs of in vitro mixing of VCP with control green fluorescent protein (GFP) or CΔ, with AT3, added in the order of the molar ratios listed (bars = 20 nm; biological repeats n = 2).