Figure 3: NKAP undergoes SUMOylation and interacts with CENP-E in mitosis.

(a) NKAP is associated with kinetochore proteins including CENP-E. HeLa cells were transfected with Flag-tagged vector or NKAP and synchronized at the indicated cell-cycle phases. Cell lysates were immunoprecipitated with anti-Flag M2 beads and the IPs were analysed by immunoblot with indicated antibodies. Phosphorylated histone H3 (Ser-10) acts as an indicator of M phase. (b) A co-IP experiment detecting the interaction between NKAP and CENP-E. HEK293T cells were co-transfected with the indicated tagged expression vectors and then treated with nocodazole for 16 h. The cell lysates with or without TurboNuclease were immunoprecipitated with anti-Flag M2 beads and analysed by immunoblot using indicated antibodies. (c) A reversed co-IP experiment detecting the interaction between NKAP and CENP-E. HEK293T cells were co-transfected with vectors expressing Flag-CENP-E_Tail and Myc-NKAP, with or without GFP-SENP2, and then immunoprecipitated with anti-Flag M2 beads and analysed by immunoblot. (d) In vivo SUMOylation assay of NKAP. HEK293T cells were transiently transfected with vectors expressing Flag-NKAP, HA-SUMO-1 or -2 together with the E2 SUMO ligase Ubc9, as indicated. After 48 h of transfection, cells were lysed and immunoprecipitated. Flag-tagged IPs were immunoblotted with the indicated antibodies. (e) In vivo SUMOylation assay of NKAP without overexpressing Ubc9 or SUMO-2 in HEK293T cells. (f) Detecting the SUMOylation of endogenous NKAP in mitotic HeLa cells. HeLa/His-SUMO-2 and parental cells were transfected with control or NKAP siRNA as indicated. His-SUMO-2 conjugates were recovered on Ni-NTA beads and analysed by immunoblot with indicated antibodies. (g) HeLa/His-SUMO-2 and parental cells were synchronized at the indicated cell-cycle phases. His-SUMO-2 conjugates were recovered and analysed by immunoblot with indicated antibodies. proM, prometaphase; Meta, metaphase.