Figure 2. Haspin binding to TOP2A CTD is... | Rockefeller University Press

Figure 2.

$Figure 2. Haspin binding to TOP2A CTD is dependent on SUMOylation and SIMs. (A) Silver stain of the pulled-down proteins using TOP2A CTD. S-tagged non-SUMOylated (CTD) and SUMOylated CTD (CTD-SUMO) through in vitro SUMOylation assay were bound to S-agarose beads and incubated with CSF XEEs for pull-down assay. After incubation with SENP2 CD, proteins were eluted with urea and precipitated with trichloroacetic acid (TCA precip.). Lanes 1 and 2 represent 5% of the S-tagged CTD and CTD-SUMO bound onto S-agarose beads as bait. Proteins in each fraction were visualized with silver stain. After elution, samples were the proteins remaining on S-agarose beads. Trichloroacetic acid–precipitated fractions were subjected to protein identification by LC-MS/MS. (B) SENP2-digested pull-down samples were analyzed by immunoblotting for Haspin. SENP2-digested S-tagged CTD was used as a loading control for the bait used in the pull-down assay. CSF lane represents 0.75% of the volume of XEEs used for each pull-down sample. CSF Haspin indicates the endogenous band found in the CSF XEEs, and Bound Haspin indicates the Haspin band in the pull-down sample. (C) Schematic representation of the primary structure of X. laevis Haspin. SIMs are located at aa 343–346 (VICL) and 364–367 (VLCL). Point mutations in each SIM are indicated in red for the disrupted SIM mutant protein (2-SIM). (D) mRNAs of GFP-tagged WT or 2-SIM Haspin were supplemented in XEEs to express Haspin-GFP, and Haspin-GFP–expressing CSF XEEs were subjected to the pull-down assay with S-tagged CTD SUMOylated (CTD-SUMO) through in vitro SUMOylation assay and bound onto S-agarose beads (middle). After SENP2-CD incubation, CTD-SUMO–bound Haspin-GFP was analyzed by immunoblotting (right). SENP2-digested S-tagged CTD was used as a loading control for the bait used in the pull-down assay. CSF XEE lanes represent 0.5% of the volume of the Haspin-GFP–expressing CSF XEEs used for each pull-down sample. (E) Quantification of pulled-down Haspin-GFP levels by CTD-SUMO, as seen in D, relative to Haspin-GFP WT levels from three independent experiments (n = 3) with levels normalized by CTD levels. Error bar represents SD. ***, P < 0.001 (Student’s t test).$

Haspin binding to TOP2A CTD is dependent on SUMOylation and SIMs. (A) Silver stain of the pulled-down proteins using TOP2A CTD. S-tagged non-SUMOylated (CTD) and SUMOylated CTD (CTD-SUMO) through in vitro SUMOylation assay were bound to S-agarose beads and incubated with CSF XEEs for pull-down assay. After incubation with SENP2 CD, proteins were eluted with urea and precipitated with trichloroacetic acid (TCA precip.). Lanes 1 and 2 represent 5% of the S-tagged CTD and CTD-SUMO bound onto S-agarose beads as bait. Proteins in each fraction were visualized with silver stain. After elution, samples were the proteins remaining on S-agarose beads. Trichloroacetic acid–precipitated fractions were subjected to protein identification by LC-MS/MS. (B) SENP2-digested pull-down samples were analyzed by immunoblotting for Haspin. SENP2-digested S-tagged CTD was used as a loading control for the bait used in the pull-down assay. CSF lane represents 0.75% of the volume of XEEs used for each pull-down sample. CSF Haspin indicates the endogenous band found in the CSF XEEs, and Bound Haspin indicates the Haspin band in the pull-down sample. (C) Schematic representation of the primary structure of X. laevis Haspin. SIMs are located at aa 343–346 (VICL) and 364–367 (VLCL). Point mutations in each SIM are indicated in red for the disrupted SIM mutant protein (2-SIM). (D) mRNAs of GFP-tagged WT or 2-SIM Haspin were supplemented in XEEs to express Haspin-GFP, and Haspin-GFP–expressing CSF XEEs were subjected to the pull-down assay with S-tagged CTD SUMOylated (CTD-SUMO) through in vitro SUMOylation assay and bound onto S-agarose beads (middle). After SENP2-CD incubation, CTD-SUMO–bound Haspin-GFP was analyzed by immunoblotting (right). SENP2-digested S-tagged CTD was used as a loading control for the bait used in the pull-down assay. CSF XEE lanes represent 0.5% of the volume of the Haspin-GFP–expressing CSF XEEs used for each pull-down sample. (E) Quantification of pulled-down Haspin-GFP levels by CTD-SUMO, as seen in D, relative to Haspin-GFP WT levels from three independent experiments (n = 3) with levels normalized by CTD levels. Error bar represents SD. ***, P < 0.001 (Student’s t test).