Importin-β partners in coIP assays from HeLa cells. (A) Synchronization protocol used for protein analysis. The mitotic shake-off sample (mostly prometaphases) was compared with cells collected at mitotic exit or in G1. (B) Coomassie blue–stained proteins in the importin-β coIP from HeLa mitotic cells (10% SDS-PAGE). Bands were excised and processed for mass spectrometry (Table S1). The inset shows an enlarged section from a parallel gel to resolve importin-β and SUMO–RANGAP1, which migrate very close. (C) RANGAP1 in cell extracts prepared either in RIPA or in nondenaturing (1D) buffer: note the reduced abundance of SUMO-conjugated forms in 1D buffer. (D) WB analysis of importin-β partners. Protein extracts were IPed using anti–importin-β antibody (top); importin-β partners are visible in the coIP pellet, noninteracting proteins are released in the supernatant (SUP). The bottom panel shows the same extract incubated with nonspecific IgG for control. (E) CoIP assays of importin-β constructs detected using GFP antibody. RANBP2 was identified in whole cell extracts (WCE) from mitotic cultures transfected with the indicated constructs (lanes 1–2 and 7–8) and in coIP together with importin-β 45−462 (lanes 3–4) and 1–876 (lanes 5–6), but not I178A/Y255A (lanes 9–10, indicated as IA/YA); the latter is functional for RAN binding. In parallel, importin-β 1–876-transfected WCE was IPed with nonspecific IgG (lanes 11–12).