EBNA3C associates with the WDR48/USP46 complex in EBNA3C-F-HA LCLs.

<p>Immunoprecipitation assay using Flag agarose to retrieve protein complexes from EBNA3C-F-HA LCLs (E3C-F-HA) is compared to flag immunoprecipiates from untagged wildtype (WT) LCLs. One percent of total cell lysate (Input) or immunoprepicitated specimens using Flag agarose (Flag IP) were separated by SDS PAGE and probed using antibodies to EBNA3C, RBPJ, CtBP1, WDR48, WDR20, USP46, or NF-kB p65.</p

WDR48 coimmunoprecipitates with RBPJ in EBV infected cells.

<p>Co-immunoprecipitation assays comparing the association of RBPJ with WDR48 in LCLs with that observed in EBV negative BL41 cells. Cell lysates were immunoprecipitated with polyclonal RBPJ sera, separated by SDS PAGE, and probed for EBNA3A, EBNA3C, WDR48, and RBPJ (as indicated).</p

Subcellular localization of USP46 complexes in LCLs.

<p>Wild type LCLs were extracted into five subcellular fractions [cytoplasm (C), membrane (M), soluble nuclear (Nu), chromatin (Ch), cytoskeleton (CS)], resolved by SDS-PAGE and probed for EBNA3 proteins, RBPJ, and components of the USP46 complex (USP46, WDR20, and WDR48). Fraction purity was assessed by probing for tubulin, BRG1, Histone H2B, and LaminB.</p

Summary of peptides identified by mass spec of tandem affinity purifications (TAP).

<p>Total number of peptides detected for each EBNA3 protein and the host proteins RBPJ, CtBP1, WDR48, WDR20, USP46 and USP12 in purified complexes is indicated. No peptides corresponding to any of these proteins were detected in the control TAP from wildtype LCLs.</p><p>Summary of peptides identified by mass spec of tandem affinity purifications (TAP).</p

ChIP assay for WDR48 at the p14ARF promoter.

<p>Chromatin immunoprecipitation (ChIP) assays were performed using antibodies for WDR48 (A) from EBNA3C-HT LCLs that were grown in the presence of 4HT (dark gray) or after 14 days of growth in the absence of 4HT (light gray). Amount of genomic DNA was measured by real time PCR using primers specific to the EBNA3C binding site in the p14^ARF promoters or sites near the EIF2AK3 and PPIA genes which bind cell transcription factors but not EBNA3C. The bar graph represents the amount of DNA precipitated relative to the amount of DNA in the corresponding input sample. The experiment shown is representative of four independent experiments and error bars indicating standard error of the mean within this experiment. Asterisk denotes that the difference in ChIP signal seen at the p14^ARF promoter is statistically significant (p = 0.01). (B) Western blot for USP46, WDR48, and tubulin levels in whole cell lysates from EBNA3CHT LCLs grown in the presence of 4HT or after 14 days of growth in the absence of 4HT.</p

Purified EBNA3 complexes exhibit DUB activity.

<p>(A) Tandem affinity purification was performed on wild type or EBNA3-F-HA expressing LCLs. Purified complexes from wild type (X), E3A-F-HA (closed circle), E3B-F-HA (closed triangle), or E3C-F-HA (closed square) LCLs were assayed for DUB activity by fluorometric assay using Ub-AMC as a substrate. (B) Western blot of purified EBNA3 complexes used in (A) demonstrating selective precipitation of tagged EBNA3 protein complexes and co-purification of RBPJ and USP46 proteins. Lysates derived from the equivalent of approximately 1.5x10⁶ cells (input), 2.4x10⁶ cells (Flag elution), or 10x10⁶ cells (HA elution) were loaded on each lane, separated by SDS PAGE, and probed with EBNA3s, Flag, EBNA3A, and HA antibodies.</p

Deletion of EBNA3A residues 920–944 disrupts WDR48 binding without affecting CtBP1 association.

<p>Co-immunoprecipitation assay to assess binding of EBNA3A mutants to WDR48 (A) and CtBP1 (B). For these assays, flag tagged full length EBNA3A (1–944), an EBNA3A CtBP1 binding mutant (mCtBP), an EBNA3A mutant lacking the C-terminal 25 residues (1–919), or vector control (pSG5) was co-transfected with Xpress-WDR48 or HA-CtBP1. Lysates were immunoprecipitated with Flag agarose (A) or HA agarose (B), separated by SDS PAGE, and probed with WDR48, RBPJ, flag, EBNA3A and HA antibodies.</p

EBNA3C associates with the WDR48/USP46 complex in EBNA3C-F-HA LCLs.

<p>Immunoprecipitation assay using Flag agarose to retrieve protein complexes from EBNA3C-F-HA LCLs (E3C-F-HA) is compared to flag immunoprecipiates from untagged wildtype (WT) LCLs. One percent of total cell lysate (Input) or immunoprepicitated specimens using Flag agarose (Flag IP) were separated by SDS PAGE and probed using antibodies to EBNA3C, RBPJ, CtBP1, WDR48, WDR20, USP46, or NF-kB p65.</p

Inability to derive USP46 null LCLs using CRISPR/ Cas9 mediated gene editing.

<p>(A) Western blot for USP46 in 721 LCLs cells transfected with a plasmid expressing either of two guide RNAs targeting different USP46 exons (CRISPR 1 or CRISPR 2) and a hygromycin resistance gene. Prior to harvesting, cells were subjected to hygromycin selection for one month (resulting in 6 clones for CRISPR 1 and 8 clones CRISPR 2). Untransfected 721 cells are also shown (WT). As a loading control, lysates were probed for beta actin (bottom panel). (B) Western blots of 293T cells that were transfected same CRISPR plasmids as in panel A and also subjected to one month of hygromycin selection. Hygromycin resistance cells were harvested and blotted for USP46 (specific band is indicated by arrowhead). Untransfected 293T cells are also shown (WT).</p

EBNA3 proteins preferentially bind the WDR48 subunit of the USP46 DUB complex.

<p>(A) Immunoprecipitation assay in 293T cells demonstrating association of flag tagged EBNA3 proteins (F-E3A, F-E3B, and F-E3C) with WDR48 (left) and WDR20 (right). (B) Immunoprecipitation assay demonstrating WDR48 cotransfection enhanced USP46 association with EBNA3s (right panel, compare lanes 3–5 with 8–10). Epitope tagged BNRF1 (F-HA-BNRF1), an EBV protein of approximately the same size as the EBNA3 is included as an additional negative control. One percent (panel A) or two percent (panel B) of the input are shown for comparison.</p