Extended Data Fig. 9: K29-linked ubiquitination is involved in mitosis.
From: K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle
Gel panels and image panels in this figure are representative of two independent experiments; n = 2. a, Western blot analysis of K29-linked ubiquitination during mitosis. HeLa cells were synchronized to prometaphase by treatment with thymidine and nocodazole. Aliquots of cells were taken and lysed at the indicated time points, followed by western blot using sAB-K29 as the primary binder. b, A diagram showing migration of the K29-linked polyubiquitination signal during the telophase of mitosis. Roughly four stages could be distinguished based on localization and morphology. c, 3D STORM images of K29-linked polyubiquitin at telophase 3 and 4. The orthographic projection image of the highlighted area in telophase 4 (bottom cyan box) along the long axis (white arrow) is shown in the upper right inset. Rendering was conducted by Visual Molecular Dynamics (VMD) with GLSL rendering, Orthographic display, and Surf drawing. Scale bars in the left images correspond to 2 μm. Scale bars in the right images correspond to 250 nm. d, Immunofluorescent staining of HeLa cells at different stages of mitosis. Costaining for K29-linked ubiquitin with α-tubulin is shown. Cells were pre-extracted before fixation. e, Immunofluorescent staining of HeLa cells with sAB-MBP. Costaining for α-tubulin is shown. Cells were pre-extracted before fixation. f, Immunofluorescent staining of HeLa cells at the telophase of mitosis. Costaining for K29-linked ubiquitin with either Aurora B or CDC27 (a negative control) is shown. Cells were pre-extracted before fixation. Scale bars in panels d-f correspond to 5 μm.
