Figure 4: SUMOylation of NKAP is required for its association with CENP-E.

(a) Left: HEK293T cells were transfected with the indicated Flag-NKAP constructs together with Myc-Ubc9 and HA-SUMO-2 for 48 h. Flag-tagged IPs were immunopurified as described in METHODS and analysed by immunoblot. Right: Schematic representation of full-length NKAP, along with its various deletion mutants. (b) Sequence alignments of NKAP C-terminal fragment (aa 276–415) from different organisms using Clustal Omega algorithm. The 14 Lys (K) mutated to Arg (R) in further assays are indicated (red filled circles). All amino acid numbers are for human NKAP. (c) In vivo SUMOylation assay for NKAP SUMOylation modification sites verification. Flag-NKAP (WT) or Flag-NKAP (14KR) mutant were expressed in HEK293T cells along with HA-SUMO-2 and Myc-Ubc9. The levels of NKAP SUMOylation were evaluated by IP of NKAP using anti-Flag M2 beads followed by anti-HA immunoblot. (d) Co-IP analysis of the association of NKAP wild type and SUMO-null mutant with CENP-E. HeLa cells were transfected with the indicated plasmids and arrested in prometaphase. The cell lysates were immunoprecipitated with anti-Flag M2 beads and analysed with the indicated antibodies. (e) SUMOylation of NKAP promotes its interaction with CENP-E. HEK293T cells were co-transfected with expression plasmids as indicated. After 48 h of transfection, the cell lysates were subjected to co-IP followed by immunoblot.