Journal: bioRxiv

Article Title: Medulloblastoma-Associated KBTBD4 Mutations Disrupt PP2A-A Orphan Quality Control

doi: 10.64898/2026.03.02.709011

Figure Lengend Snippet: A. PP2A subunits PP2A- 3×Flag Aα/ Strep Cα/B55α were expressed as individual proteins, in PP2A subcomplexes PP2A-AC/AB/CB, and as the heterotrimeric PP2A-ACB holoenzyme in insect cells. PP2A extracts were mixed as indicated with 6×His-MBP KBTBD4 extract and affinity purified to test for binding of KBTBD4 to PP2A subunits and subcomplexes. B. Purified PP2A subunits and (sub-)complexes were ubiquitylated in vitro with purified KBTBD4, CUL3 NEDD8 -RBX1, UBE1, UBE2D3 and fluorescently labeled ubiquitin. Representative result of n=2 independent experiments. C. Cellular extracts from HEK293T cells and Δ KBTBD4 cell lines #1and #2 were separated over a 5-20% sucrose gradient by ultracentrifugation. Fractions were probed for indicated proteins by western blot. Representative result of n=2 experiments.

Article Snippet: Ubiquitylation assays were carried out in 20 μL reactions with final concentrations of 1 μM PP2A (PP2A-A or in indicated complex), 0.1 μM UBE1 (R&D Systems, E-304), 0.2 μM UBE2D3 (R&D Systems, E2-627), 0.5 μM KBTBD4, 0.4 μM CUL3 NEDD8 -RBX1 (CRL), 7.5 μM ubiquitin IRdye680 (gift from Jake Aguirre, as published in ( )), 5 mM ATP, 2.5 mM MgCl 2 , in a buffer of 20 mM HEPES pH 7.4, 150 mM NaCl and 1 mM DTT.

Techniques: Affinity Purification, Binding Assay, Purification, In Vitro, Labeling, Ubiquitin Proteomics, Western Blot

Journal: bioRxiv

Article Title: Medulloblastoma-Associated KBTBD4 Mutations Disrupt PP2A-A Orphan Quality Control

doi: 10.64898/2026.03.02.709011

Figure Lengend Snippet: A. Flag purification of transiently expressed wild-type KBTBD4 3×Flag and medulloblastoma hotspot mutants to probe for binding to 2×HA PP2A-A. B. PP2A-A stability was monitored by flow cytometry through transient expression of the dual fluorescence reporter in HEK293T cells and Δ KBTBD4 #1 with co-expression of KBTBD4 WT or the MB mutant KBTBD4 PR construct. C. Quantification of the stability of PP2A-A as monitored by flow cytometry after expression of the dual fluorescence reporter and indicated KBTBD4 constructs (WT, MB mutant PR, CUL3-binding mutant C3). Data was measured at least in triplicates, and the median fluorescence signal ratio of GFP over mCherry was normalized to HEK293T control. D. Ubiquitin conjugates were purified under denaturing conditions from Δ KBTBD4 #1 cells, virally transduced to rescue with indicated KBTBD4 constructs, and transiently transfected to express His-tagged ubiquitin and myc PP2A-A. E. Purified PP2A-A was ubiquitylated in vitro with purified KBTBD4 (WT and MB cancer mutant PR), CUL3 NEDD8 -RBX1, UBE1, UBE2D3 and fluorescently labeled ubiquitin. Representative result of n=2 independent experiments. F. Flag purification of transiently expressed 3×Flag PP2A-A (wild-type and cancer mutants P179R and R183W) to probe for binding to KBTBD4 3×HA . G. Purified PP2A-A (WT, and cancer mutants P179R and R183W) was ubiquitylated in vitro with purified KBTBD4, CUL3 NEDD8 -RBX1, UBE1, UBE2D3 and fluorescently labeled ubiquitin. Representative result of n=2 independent experiments. H. Quantification of the stability of wild-type PP2A-A and indicated mutants as monitored by flow cytometry after viral transduction of a dual fluorescence reporter. Data representative of n=3 independent experiments.

Article Snippet: Ubiquitylation assays were carried out in 20 μL reactions with final concentrations of 1 μM PP2A (PP2A-A or in indicated complex), 0.1 μM UBE1 (R&D Systems, E-304), 0.2 μM UBE2D3 (R&D Systems, E2-627), 0.5 μM KBTBD4, 0.4 μM CUL3 NEDD8 -RBX1 (CRL), 7.5 μM ubiquitin IRdye680 (gift from Jake Aguirre, as published in ( )), 5 mM ATP, 2.5 mM MgCl 2 , in a buffer of 20 mM HEPES pH 7.4, 150 mM NaCl and 1 mM DTT.

Techniques: Purification, Binding Assay, Flow Cytometry, Expressing, Fluorescence, Mutagenesis, Construct, Control, Ubiquitin Proteomics, Transfection, In Vitro, Labeling, Transduction