Figure 6. Dynamic ubiquitination of Oct1 during mitosis.

(A) HeLa cells were blocked in G1 with thymidine, released and arrested in mitosis using 0.1 µM nocodozole as described [22]. Top panels show Western blot with K11-Ub and pan-Oct1 antibodies, and GAPDH antibodies as a loading control, from cells arrested with nocozole or following 2 hr release from nocozole. For the bottom panels, samples were immunoprecipitated using anti-Oct1^pS335 antibodies, and probed with antibodies against pan-Ub, K11-Ub and pan-Oct1. H.C. = immunoglobulin heavy chain. (B) IF images of HeLa mitoses stained with Oct1^pS335 and K11-Ub antibodies. Two metaphase, telophase and late cytokinesis examples are shown. Merged images from the two latter cases also show detail of the midbody structure (inset). (C) HeLa cells were arrested with nocodozole (0.5 µM) for 18 hr, and then incubated with MG132 for a further 6 hr. Whole cell extracts were prepared and subjected to Western blotting using Oct1^pS335 and K11-Ub antibodies. (D) HeLa cells were treated with MG132, fixed and subjected to IF using K11-Ub and Oct1^pS335 antibodies. Detail of metaphase cells is shown. (E) Whole cell extracts from HeLa cells arrested as above were immunoprecipitated with anti-Oct1^pS335 antibodies and Western blotted using pan-Ub, K11-Ub or pan-Oct1 antibodies. H.C. = immunoglobulin heavy chain. (F) Nocodozole-arrested HeLa whole cell extracts were immunoprecipitated using Oct1^pS335 antibodies and probed using pan-Oct1 or APC1 (AbCam). (G) Verification of Cdh1 knockdown. HeLa cells were transfected with Cdh1 siRNAs for 48 hr, after which cells were treated with 0.5 mM nocodozole for 18 hr. A Western blot is shown using Cdh1 antibodies. GAPDH is shown as a loading control. (H) HeLa cells were transfected with Cdh1 siRNAs. 24 hr-post transfection, cells were treated with 0.5 µM nocodozole. 42 hr-post transfection, cells were treated with MG132 for 6 hr. Whole cell extracts were prepared after 48 hr. (I) siRNA-transfected HeLa cells as in (H) were immunoprecipitated using Oct1^pS335 antibodies and Western blotted using pan-Ub or K11-Ub antibodies.