Fig 1: Kinetochore recruitment of Ska1 involves its N terminal disordered loop.A, cartoon representation of Ska complex showing the domains of individual Ska proteins (Ska1, Ska2, and Ska3). Ska1, 2, 3 are labeled with different colors. Microtubule-binding C-terminal domains of Ska1 and Ska3 are labeled as MTBD. The N-terminal oligomerization domain is labeled as OD. B, schematic representation of GFP-tagged full length Ska1 and Ska1 Δ loop constructs. C, representative immunofluorescence confocal microscopy images of HeLa cells transfected with control siRNA, Ska1 siRNA, Ska1 siRNA+ siRNA-resistant Ska1-GFP (48 h), and Ska1 siRNA+ siRNA-resistant Ska1 Δloop-GFP (48 h). Control and Ska1 siRNA only–treated cells were immunostained with rabbit polyclonal Ska1 antibody (green), and EB1 was stained with rat monoclonal EB1 antibody (red). Ska1-GFP or Ska1 Δloop-GFP–expressed cells were stained with polyclonal EB1 rabbit antibody. The GFP channels were imaged directly. Microtubules were stained with α-tubulin mouse monoclonal antibody in all cases (violet). DNA was stained with DAPI (shown in white). Scale bar represents 5 μm. D, Western blot images of cell lysates of Ska1 siRNA-, Ska1 siRNA + Ska1-GFP, and Ska1 siRNA + Ska1 Δloop-GFP–treated cells showing expression levels of the exogenous Ska1 proteins with simultaneous depletion of endogenous Ska1. E, plot showing percentage of mitotic cells with misaligned chromosomes in Ska1 siRNA-, Ska1 siRNA + Ska1-GFP-, and Ska1 siRNA + Ska1 Δloop-GFP–treated conditions. Data are mean ± SEM. ∗∗∗∗ represents p< 0.0001. F, HeLa cells in Ska1 siRNA + Ska1-GFP or Ska1 siRNA + Ska1 Δloop-GFP–treated condition were imaged for localization of the GFP-fused Ska1 proteins at the kinetochore (KT). Insets 1 and 2 represent GFP-tagged Ska1 proteins and CENP-A, respectively. EB1 was stained with rabbit EB1 antibody, and CENP-A was stained with mouse monoclonal CENP-A antibody. DNA was stained with DAPI. The scale bars in the main and inset figures are 5 μm and 1 μm, respectively. G, plot showing the intensity of Ska1-GFP versus Ska1 Δloop-GFP at individual KTs in HeLa cells. Data are mean ± SEM. ∗∗∗∗p< 0.0001 (n = 3). Approximately, 100 KTs in each of the three experiments were measured.
Fig 2: Hypoxia-induced SKA3 interacted with HIF-1a and hindered its degradation in a UPS-dependent manner. A Western bolt analysis was used to detect the expression of HIF-1a in SKA3 knockdown and overexpression cells under hypoxic conditions. B Immuno-fluorescence technique (IF) verified the co-location between SKA3 and HIF-1a. C Co-immunoprecipitation with anti-SKA3, anti-HIF-1a, and anti-IgG was performed to identify the binding between SKA3 and HIF-1a under hypoxic and normoxic conditions. D Cells with SKA3 knockdown or overexpression were treated with cyclohexamide (CHX) for indicated time to determine the half-life of HIF-1a protein. E Western blotting analysis was used to detect the expression of SKA3 and HIF-1a under normoxic (N) or hypoxic conditions (H) with or without MG132 in SKA3 knockdown and overexpression CCA cells. F-G Lysates from SKA3 knockdown and SKA3 overexpression CCA cells treated with MG132 and transfected with HA-Ub or HA-Ub lys48 alone under hypoxic conditions, and then the level of ubiquitinated HIF-1a was analysed
Fig 3: Hypoxia-induced SKA3 protects CCA cells against the cytotoxic effects of gemcitabine. A CCA cells with SKA3 overexpression were treated with gemcitabine at different dose under hypoxic conditions, then cell viability was examined after 48 h treatment. C CCA cells with SKA3 overexpression group and NC group were treated with gemcitabine at dose of 500 nM/mL under hypoxic conditions, then clone formation assays (B) and DNA damage detection assays (C) were performed. D-F The efficacy of gemcitabine in vivo was determined in nude mice injected with SKA3 overexpression QBC939 cells. G Representative images of H&E staining in tumour derived from DMSO or gemcitabine-treated nude mice with SKA3 overexpression group and NC group cells inoculation (scale bar: 50μm). *P < 0.05
Supplier Page from Abcam for Anti-SKA3 antibody - C-terminal