Journal: bioRxiv

Article Title: Chromosome segregation fidelity is controlled by small changes in phospho-occupancy at the kinetochore-microtubule interface

doi: 10.1101/2021.02.16.431549

Figure Lengend Snippet: A) Immunofluorescence images from an asynchronous population of HeLa cells transfected with control or a pool of siRNA against B56 α-ε and then stained for Hec1 pS44, Hec1 and DAPI. The Hec1 and Hec1 pS44 channels were adjusted evenly for brightness and contrast. The DAPI channel was adjusted independently. The scale bars for main images are 10 μm and 1 μm for insets. Representative images of 3 independent experiments are shown. B) Quantification of the relative kinetochore intensities from the conditions in panel (A). The condition with the lowest level of pS44 was set to 1 and the other conditions shown as fold-changes. 25 kinetochores were quantified from each of 20 cells for each of 3 independent repeats. Error bars indicate the mean +/- SEM. **** denotes p < 0.0001. C) Immunofluorescence images of cells prepared as in panel (A) except stained for pT31 instead of pS44. D) Quantification of the relative kinetochore intensities from the conditions in panel (C). The condition with the lowest level of pT31 was set to 1 and the other conditions shown as fold-changes. 25 kinetochores were quantified from each of 20 cells for each of 3 independent repeats. Error bars indicate the mean +/- SEM. **** denotes p < 0.0001. E) Immunofluorescence images from an asynchronous population of HeLa cells transfected with control or a pool of siRNA against B56 α-ε. The media was then changed for media at 4°C, and the cells placed at 4°C for 15 minutes prior to fixation and staining with antibodies against ACA and α Tubulin. Representative images of 3 independent experiments are shown. F) Quantification of the relative intensity of α Tubulin. The levels of the control condition were set to 1 and the other condition shown as a fold-change. At least 50 cells per condition from each of 3 independent experiments were quantified. Error bars indicate the mean +/- SEM. **** denotes p < 0.0001. G) Cartoon illustration showing the kinase and phosphatase network regulating Hec1 phosphorylation. In early prometaphase, cells without k-MT attachments have elevated levels of fractional phosphorytion on several Hec1 sites that are phosphorylated by Aurora kinases (green circles) or Cdk1-Cyclin B1 kinase (magenta circles). When lateral k-MT attachments occur, the levels of phosphorylation generally decrease. Finally, when end-on k-MT attachments occur, levels of phosphorylation decrease further. Phosphorylation levels at Hec1 S4,5,8 and S62 are speculative or based on partial data coverage.

Article Snippet: The following antibodies were used for immunofluorescence (IF) and/or immunoblotting (IB): ACA (Geisel School of Medicine; IF at 1:2000), Hec1 (Santa Cruz C-11; IF,IB at 1:1000), PP2A B56 Alpha (MyBioSource.com MBS8524809; IB at 1:500), PP2A B56 Beta (Santa Cruz E-6; IB at 1:500), PP2A B56 Gamma (Santa Cruz A-11; IB at 1:500), PP2A B56 Delta (Santa Cruz H-11; IB at 1:500), PP2A B56 Epsilon (Aviva Systems Biology RP56694_P050; IB at 1:500), Nuf2 (Santa Cruz E-6; IB at 1:1000), phospho-Hec1 Ser44 (gift of Jennifer DeLuca; IF at 1:500), GAPDH (Santa Cruz G-9; IB at 1:1000), phospho-Histone H3 Ser10 (Cell Signaling Technologies #9701; IB at 1:2000), FLAG M2 (Sigma-Aldrich; IF and IB at 1:1000), Cyclin A2 (Genetex GT2547; IF at 1:1000), Cyclin B1 (Santa Cruz GNS1; IF at 1:500), α-Tubulin DM1α (Sigma-Aldrich; IF at 1:4000).

Techniques: Immunofluorescence, Transfection, Control, Staining, Phospho-proteomics

Journal: Scientific Reports

Article Title: Ganglioside GQ1b ameliorates cognitive impairments in an Alzheimer’s disease mouse model, and causes reduction of amyloid precursor protein

doi: 10.1038/s41598-019-44739-6

Figure Lengend Snippet: GQ1b decreases tau phosphorylation accompanied by increased levels of phospho-GSK3β in 3xTg-AD mice. ( A ) Western blotting and ( B ) its quantification revealed that GQ1b administration into the hippocampus of 3xTg-AD mice significantly reduced phospho-tau (T181, T205, T231) levels compared to aCSF-infused 3xTg-AD mice. ( C ) Western blotting and ( D ) its quantification showed that GQ1b infusion rescued the decrease in phospho-GSK3β levels without affecting expression of a representative tau phosphatase, PP2A. Western blotting band intensity was quantified by densitometry analysis on pTau, Tau, pGSK3β, GSK3β, PP2A, and GAPDH bands. Western blots shown represent typical results from three independent experiments, and the graphs show data from three independent experiments and are expressed as mean values ± SD. * p < 0.05, ** p < 0.01 vs. WT + aCSF group, # p < 0.05, ## p < 0.01 vs. AD + aCSF group.

Article Snippet: Proteins were transferred onto nitrocellulose membranes (Whatman, Milford, MA, USA) and then blocked by 5% skim milk (BD Bioscience, Regilait, Saint-Martin-Belle-Roche, France) for 1 h. The following primary antibodies were incubated at 4 °C overnight to probe proteins: rabbit anti-BDNF (1:2000, Alomone Labs, Jerusalem, Israel), rabbit-anti presenilin 1 (1:2000, Cell Signaling, Beverly, MA, USA), rabbit anti-pGSK3β (1:2000, Cell Signaling), rabbit anti-GSK3β (1:2000, Cell Signaling), mouse anti-Tau (1:3000, Cell Signaling), rabbit anti-cleaved PARP (1:2000, Cell Signaling), rabbit anti-ADAM10 (1:1000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-Neprilysin (1:1000, Santa Cruz Biotechnology), rabbit anti-β-actin (1:5000, Bethyl Laboratories, Montgomery, TX, USA), rabbit anti-pTau T181, T205, T231 (1:1000, 1:1000, 1:1000, ThermoScientific, Rockford, IL, USA), rabbit anti-IDE (1:1000, Abcam, Cambridge, UK), mouse anti-BACE1 (1:2000, R&D Systems, Minneapolis, MN, USA), mouse anti-GAPDH (1:5000, Millipore, Temecula, CA, USA), mouse anti-PP2A (1:2000, AbFrontier, Seoul, Korea), and mouse anti-APP/Aβ (1:5000, 6E10, Covance Research Products, Dedham, MA, USA).

Techniques: Western Blot, Expressing