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. 2021 Jun 15;220(7):e202008037. doi: 10.1083/jcb.202008037

Figure 3.

Figure 3.

SH3BP5 and SH3BP5L are Tankyrase substrates. (A) Interaction network of significant SH3BP5 interactors identified by BioID. The interaction network was created using Cytoscape and grouped by biological function using the Gene Ontology database. Hexagon size indicates the spectrum sum of each BioID run. Solid black lines indicate known protein interactions imported from the GeneMANIA plugin and dotted lines represent new interactions identified by BioID. (B) Protein domain maps of SH3BP5 and SH3BP5L. Green boxes represent the conserved four α-helices predicted by the PSIPRED server. (C and D) HEK293T lysates coexpressing Flag-SH3BP5 or Flag-SH3BP5L and Myc-TNKS or Myc-TNKS2 were immunoprecipitated with Myc (9E10) antibody and immune complexes were subjected to Western blot analysis and probed for Flag and Myc expression. Whole-cell lysates were examined for Flag and Myc expression and α-tubulin serves as a loading control. (E and F) Lysates from HEK293T coexpressing Flag-SH3BP5 or Flag-SH3BP5L or respective individual or combined TBM deletion mutants (SH3BP5 - Δ1; Δ2; Δ1,2 or SH3BP5L - Δ1; Δ2; Δ3; Δ1,2; Δ1,3; Δ2,3; Δ1,2,3) with Myc-TNKS or Myc-TNKS2. Lysates were immunoprecipitated with Flag antibody and complexes were subjected to Western blot analysis for Myc and Flag expression. Whole-cell lysates were examined for Myc and Flag expression and α-tubulin serves as a loading control. (G and H) HEK293T were coexpressed with Flag-SH3BP5 or Flag-SH3BP5L with Myc-TNKS or Myc-TNKS2. Lysates were immunoprecipitated with Flag antibody and immune complexes were washed and incubated with 10 µM Biotin-NAD for 1 h. This mixture was subjected to Western analysis and PARylation was detected by streptavidin-HRP. Complexes were also probed for Myc and Flag expression while whole-cell lysates were examined for Myc and Flag and α-tubulin serves as a loading control. * indicates TNKS/TNKS2 bands and → indicates SH3BP5 bands. (I and J) Same as G and H, but cells were treated with and without 5 µM TNKS656 inhibitor for 24 h before lysis. * indicates TNKS/TNKS2 bands and → indicates SH3BP5/SH3BP5L bands. TBM, Tankyrase binding Motif. Molecular weights (kD) for blots are indicated on the left of each panel.