Journal: Nature Chemical Biology

Article Title: Tau phosphorylation impedes functionality of protective tau envelopes

doi: 10.1038/s41589-025-02122-9

Figure Lengend Snippet: a . Schematics of lysate preparation from HEK293T cells transfected with GFP-tau (HEK lysate, top, cyan) or GFP-tau and Cdk5/p25 (Cdk5 lysate, bottom, magenta). b . Western blot of HEK293T cells transfected with GFP-tau (HEK lysate) or GFP-tau and Cdk5/p25 (Cdk5 lysate) using total tau antibody (tau-5, top panel) and phospho-specific antibodies (AT8, AT180 and pS404). Three independent samples per condition were analyzed from one Western blot. c . Total tau levels (tau-5 intensity) of HEK (cyan) and Cdk5 lysate (magenta), presented as mean ± s.d. (n = 3, 3 independent samples). d . Quantification of relative phosphorylation levels, normalized to total tau. Mean HEK lysate (cyan) was set to 1 and compared to Cdk5 lysate levels (magenta), presented as mean ± s.d. (n = 3, 3 independent samples). e . Multichannel fluorescence micrographs of HEK (top, cyan) and Cdk5 lysate (bottom, magenta) on taxol-stabilized microtubules (black, IRM). Estimated tau concentrations: 30 and 40 nM, respectively. Scale bars: 2 μm. f . Percentage of microtubules covered by tau envelopes after addition of HEK (cyan) or Cdk5 lysate (magenta), presented as mean ± s.d. (n = 8, 8 independent experiments). Two-sided t-test, p = 4.3821*10-10. g . Fluorescence micrographs of GFP-tau (left) and mScarlet-tubulin (right) in U-2 OS cells in interphase (cyan) or in mitosis (magenta). h . Pearson’s R correlation coefficient between GFP-tau and mScarlet-tubulin signal, presented as mean ± s.d. (n = 25, 20 cells in 3, 3 independent experiments). Two-sided t-test, p = 1.4224*10-7. i . Coefficient of variation (CoV) of tau signal in the whole cell reflecting the difference between tau signal on microtubules and in cytoplasm. CoV for interphase (cyan) and mitotic (magenta) cells, presented as mean ± s.d. (n = 25, 19 cells in 3, 3 independent experiments). Two-sided t-test, p = 5.2528*10-20. j . Mean tau signal in interphase (cyan) and mitotic (magenta) cells as a measure of the expression level, presented as mean ± s.d. (n = 25, 19 cells in 3, 3 independent experiments). Two-sided t-test, p = 0.099936753. Source data for b , c , d , f , h , i and j is available with this manuscript. Stars for statistical tests represent: *** p < 0.001.

Article Snippet: Biotin-labeled tubulin was purchased from Cytoskeleton (T333P) and diluted 1:50 with unlabeled porcine brain tubulin to obtain biotin-labeled tubulin mix for surface-immobilization assays using biotin antibodies.

Techniques: Transfection, Western Blot, Phospho-proteomics, Fluorescence, Expressing

Journal: Nature Chemical Biology

Article Title: Tau phosphorylation impedes functionality of protective tau envelopes

doi: 10.1038/s41589-025-02122-9

Figure Lengend Snippet: a . Fluorescence micrographs of IMCD-3 cells before elevated-pH treatment (top), at t = 0 min after elevated-pH treatment (middle) and t = 9 min after elevated-pH treatment (bottom). Types of cells subjected to the pH treatment: control GFP-tau cells (tau, cyan), GFP-tau-∆N cells (tau-∆N, orange), and GFP-tau + Cdk5/p25 cells (tau-Cdk5, magenta). Left panels show the mScarlet-tubulin signal, right panels the GFP-tau signal. Scale bars: 10μm. b . Density of tau on microtubules before elevated-pH treatment compared to density of tau in the cytoplasm, to give a measure of the binding affinity of tau in the different cells. Data is presented as mean ± s.d. (n = 25 cells for each group in 4 independent experiments). Two-sided t-test p-values (left-to-right): p = 3.26*10-8, 0.0594, 5.89*10-4. c . Mean intensity of tau before elevated pH-treatment, as an indication of the expression level, presented as mean ± s.d. (n = 25 cells for each group in 4 independent experiments). Two-sided t-test p-values (left-to-right): p = 0.0658, 0.8901, 0.0774. Source data for b and c is available with this manuscript. Stars for statistical tests represent: * p < 0.05, *** p < 0.001.

Article Snippet: Biotin-labeled tubulin was purchased from Cytoskeleton (T333P) and diluted 1:50 with unlabeled porcine brain tubulin to obtain biotin-labeled tubulin mix for surface-immobilization assays using biotin antibodies.

Techniques: Fluorescence, Control, Binding Assay, Expressing

Journal: Nature Chemical Biology

Article Title: Tau phosphorylation impedes functionality of protective tau envelopes

doi: 10.1038/s41589-025-02122-9

Figure Lengend Snippet: a . Change in length of glycerol-stabilized microtubules immediately after buffer change (left) and 9 min later (right). Buffer was switched from pH 7.4 to pH 8.4 (purple), or pH 7.4 to pH 7.4 (control, grey). Data is presented as mean + s.d. (n = 3, 3 independent experiments with N = 19, 16 microtubules). Two-sided t-test p-values (left to right): p = 0.9417, 0.8982. b . Fluorescence micrographs of IMCD-3 cells before elevated pH-treatment (left), and right after elevated pH-treatment (t = 0 min, right). The generated mask from the microtubule signal (mScarlet-tubulin) and corresponding tau signal (GFP-tau, control) is shown next to the image (Methods). Scalebars: 2μm. c . Length of microtubule signal (grey) or tau signal (cyan) right after elevated pH-treatment normalized to the length before pH treatment, presented as mean ± s.d. (n = 13 areas in 7 cells). Two-sided t-test, p = 1.17238*10-8. d . Coefficient of variation (CoV) of GFP-tau signal (top) or mScarlet-tubulin signal (bottom) measured along the microtubule lattice in GFP-tau (cyan), GFP-tau-∆N (orange), and GFP-tau-Cdk5 (magenta) cells. CoV was measured at 3 timepoints: before elevated-pH treatment (left), at t = 0 min after treatment (middle), and at t = 9 min after treatment (right). Data is presented as mean ± s.d. (CoV of tau, n = 25, 25, 25 cells; CoV of tubulin, n = 25, 25, 25 cells). Two-sided t-test p-values (CoV tau left-to-right): p = 0.0032423, 0.002724, 0.541674, 0.035050, 0.446571, 0.001353, 0.279565, 0.017556, 0.002562; (CoV tubulin left-to-right): p = 0.147458, 0.044724, 0.395936, 0.012266, 0.539248, 0.017755, 0.733709, 0.040452, 0.125798. Source data for a , c and d is available with this manuscript. Stars for statistical tests represent: * p < 0.05, ** p < 0.01.

Article Snippet: Biotin-labeled tubulin was purchased from Cytoskeleton (T333P) and diluted 1:50 with unlabeled porcine brain tubulin to obtain biotin-labeled tubulin mix for surface-immobilization assays using biotin antibodies.

Techniques: Control, Fluorescence, Generated

Journal: Nature Chemical Biology

Article Title: Tau phosphorylation impedes functionality of protective tau envelopes

doi: 10.1038/s41589-025-02122-9

Figure Lengend Snippet: a , Fluorescence micrographs of IMCD-3 cells expressing mScarlet–tubulin and GFP–tau in control cells (tau; cyan) at t = 0 min (left), t = 5 min (middle) and t = 9 min (right) after elevated-pH treatment. Corresponding linescans are shown below the micrographs. Different microtubules were selected at different time points because of dynamic microtubule behavior. Scale bars, 1 µm. b , Tau density in patches of GFP–tau control cells (tau; cyan) and GFP–tau-Cdk5/p25 cells (tau-Cdk5; magenta), normalized to the tau density along the microtubules before treatment (dark blue). In GFP–tau-∆N cells (tau 242–441; orange), normalized tau density is measured along the microtubule lattice (no patches are visible in tau-∆N cells). Data are presented as the mean (bar) and all individual data points ( n = 10 microtubules). c , Fluorescence micrographs of mScarlet–tubulin and GFP–tau signal in tau-∆N cells (orange) at t = 0 min (left), t = 5 min (middle) and t = 9 min (right) after elevated-pH treatment. Corresponding linescans are shown below the micrographs. Different microtubules were selected at different time points because of dynamic microtubule behavior. Scale bars, 1 µm. d . Fluorescence micrographs of mScarlet–tubulin and GFP–tau signal in tau-Cdk5 (magenta) at t = 0 min (left), t = 5 min (middle) and t = 9 min (right) after elevated-pH treatment. Corresponding linescans are shown below the micrographs. Different microtubules were selected at different time points because of dynamic microtubule behavior. Scale bars, 1 µm. e , Time trace of tau density on microtubules after elevated-pH treatment normalized to the tau density on microtubules before the treatment in GFP–tau cells (tau; cyan) and GFP–tau-Cdk5/p25 cells (tau-Cdk5; magenta). Data are presented as the mean ± s.d. at each time point ( n = 25 cells) and fitted with an exponential curve . Exponential time constant was 1.3 min for tau cells and 2.5 min for tau-Cdk5 cells . * P < 0.05 and *** P < 0.001. Int., intensity.

Article Snippet: Biotin-labeled tubulin was purchased from Cytoskeleton (T333P) and diluted 1:50 with unlabeled porcine brain tubulin to obtain biotin-labeled tubulin mix for surface-immobilization assays using biotin antibodies.

Techniques: Fluorescence, Expressing, Control

Journal: Nature Chemical Biology

Article Title: Tau phosphorylation impedes functionality of protective tau envelopes

doi: 10.1038/s41589-025-02122-9

Figure Lengend Snippet: a , Fluorescence micrographs of IMCD-3 cells expressing katanin–GFP (top), fixed 12 h after transfection and stained for tubulin (bottom). In combination with katanin, cells express full-length mCherry–tau (+tau; cyan), mCherry–tau-∆N (+tau-∆N; orange), mCherry–tau and Cdk5/p25 (+tau-Cdk5; magenta) or Cdk5/p25 in the absence of tau (+Cdk5; light pink). Cells expressing katanin are marked with yellow dotted lines and cells not expressing katanin (nontransfected cells) are marked with white dotted lines. Scale bars, 5 µm. Experiments were performed three times for each cell type, yielding similar results. b , Relative tubulin density 12 h after transfection, presented as the mean ± s.d. ( n = 132, 131 and 132 cells in six independent experiments or 60, 60 and 72 cells in three independent experiments). c , Correlation of the relative tubulin density ( y axis) compared to the relative katanin density ( x axis). Correlation coefficients were −0.49 (katanin only; yellow), −0.25 (+tau; cyan), −0.57 (+tau-Cdk5; magenta) and −0.54 (+Cdk5; light pink).

Article Snippet: Biotin-labeled tubulin was purchased from Cytoskeleton (T333P) and diluted 1:50 with unlabeled porcine brain tubulin to obtain biotin-labeled tubulin mix for surface-immobilization assays using biotin antibodies.

Techniques: Fluorescence, Expressing, Transfection, Staining

Journal: Nature Chemical Biology

Article Title: Tau phosphorylation impedes functionality of protective tau envelopes

doi: 10.1038/s41589-025-02122-9

Figure Lengend Snippet: a . Relative tau density in cells expressing Katanin-GFP in combination with mCherry-tau ( + tau, cyan) or mCherry-tau-∆N ( + tau-∆N, orange). Tau densities were set to 1 for tau cells and compared to the densities of tau-∆N cells, presented as mean ± s.d. (n = 75, 75 cells in 3, 3 independent experiments). Two-sided t-test, p = 3.3153*10-6. b . Correlation of the relative tubulin density (y-axis) compared to the relative katanin density (x-axis). Correlation coefficients of ‘katanin only‘ is -0.008 (yellow), of ‘+ tau‘ is -0.001 (cyan), and of ‘+ tau-∆N‘ is -0.006 (orange). c . Fluorescence micrographs of IMCD-3 cells co-transfected with mCherry in combination with Cdk5/p25 to visualize transfected cells (‘Cdk5/p25 alone’, left panels) or with mCherry in combination with an empty vector (control, ‘mCherry alone’, right panels). Cells were fixed and stained 12 hours after transfection with anti-tubulin (grey, middle panels) and anti-p25 antibodies (green, bottom panel). Cells expressing mCherry are marked with a yellow dotted line. Scale bars: 5 µm. d . Relative tubulin density in IMCD-3 cells transfected with mCherry in combination with Cdk5/p25 (‘Cdk5/p25 alone’, light pink) or mCherry and empty vector (‘mCherry alone’, grey). Data is presented as mean ± s.d. (n = 63, 67 cells in 2, 2 independent experiments). Two-sided t-test p = 0.46689. e. Relative tau density in cells expressing mCherry-tau (tau, cyan) or mCherry-tau + Cdk5/p25 (tau-Cdk5, magenta). Tau densities were set to 1 for tau cells and compared to the densities of tau-Cdk5 cells (mean ± s.d., n = 75, 75 cells in 3, 3 independent experiments). Two-sided t-test, p = 0.319199. Source data for a , b , d and e is available with this manuscript. Stars for statistical tests represent: *** p < 0.001.

Article Snippet: Biotin-labeled tubulin was purchased from Cytoskeleton (T333P) and diluted 1:50 with unlabeled porcine brain tubulin to obtain biotin-labeled tubulin mix for surface-immobilization assays using biotin antibodies.

Techniques: Expressing, Fluorescence, Transfection, Plasmid Preparation, Control, Staining

Journal: eLife

Article Title: An Eya1-Notch axis specifies bipotential epibranchial differentiation in mammalian craniofacial morphogenesis

doi: 10.7554/eLife.30126

Figure Lengend Snippet:

Article Snippet: Antibodies for western blot were: anti-Notch1 (1/300, sc-6014, Santa Cruz), anti-Eya1 (1/500, H00002138-A01, Abnova), anti-Hes1 (1/500, ab71559, Abcam), anti-Hey1 (1/500, ab22614, Abcam), anti-Gapdh (1/10000, ab8245, Abcam), anti-Notch1 1ICD (V1744) (1/500, 2421S, Cell Signaling), anti-Flag (1/4000, F7425, Sigma-Aldrich, Missouri, USA), anti-Myc (9E10) (1/1000, sc-40, Santa Cruz), anti-α-tubulin (1/5000, AA4.3-s, DSHB), anti-HA (1/1000, sc-805, Santa Cruz), anti-Phospho-Threonine-Proline (1/3000, 9391S, Cell Signaling), anti-Phospho-Serine-Proline-Proline motif (1/1000, 14390, Cell Signaling), and anti-Phospho-Tyrosine (1/1000, PY99, Santa Cruz).

Techniques: Recombinant, Transfection, Protease Inhibitor, In Situ, Sequencing, Clone Assay, In Situ Hybridization, Construct, Generated, Plasmid Preparation, Mutagenesis, Software

Journal: Cell Stem Cell

Article Title: Stage-Specific Transcription Factors Drive Astrogliogenesis by Remodeling Gene Regulatory Landscapes

doi: 10.1016/j.stem.2018.09.008

Figure Lengend Snippet:

Article Snippet: Immunoblotting was performed as described previously ( ) using following antibodies: mouse anti-GFAP (1:1000, G3893, Sigma), rabbit anti-CX43 (1:000, ab11370, Abcam), rat anti-ASCL1 (1:1000, MAB2567, R&D systems), rabbit anti-NES (1:1000, 839801, BioLegend), rabbit anti-OLIG2 (1:1000, AB9610, Milipore), rabbit anti-NFIA (1:1000, ab41851, Abcam), rabbit anti-RUNX2 (1:1000, sc-10758 X, Santa Cruz), rabbit anti-GAPDH (1:1000, ab9484, Abcam) and mouse anti-FLAG (1:1000, F1804, Sigma).

Techniques: Recombinant, Protease Inhibitor, Ligation, SYBR Green Assay, Plasmid Preparation, Software, Microscopy

Journal: The Journal of Cell Biology

Article Title: Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1

doi: 10.1083/jcb.200810159

Figure Lengend Snippet: Chk1 levels are reduced at centrosomes in both MCPH1- and PCNT-deficient cells. (A) Normal, MCPH1 427insA , and PCNT 3109G>T LBCs as well as U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA were costained with mouse anti-Chk1 (red) and rabbit anti–γ-tubulin (green) antibodies and analyzed by confocal microscopy. Bar, 10 µm. (B) The mean percentages of cells with centrosomal colocalization of γ-tubulin and Chk1 are indicated. Error bars represent the standard deviation after combining the results of three different experiments. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 9 × 10 −6 (MCPH1 427insA ); P = 0.00003 (PCNT 3109G>T ). (C) Quantification of pixel intensity profiles constructed from optically sectioned ( z axis) fluorescence images of control, MCPH1 427insA , and PCNT 3109G>T LBCs. Error bars represent standard deviations from the analysis of 100 cells. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 1.4 × 10 −7 (MCPH1 427insA ); P = 9.8 × 10 −12 (PCNT 3109G>T ). (D) Loss of Chk1 protein in isolated centrosome preparations. Immunoblots were performed on three sucrose gradient fractions of centrosome preparations (left) and whole cell lysates (right) as an input control from normal, MCPH1 427insA , and PCNT 3109G>T LBCs using antibodies against Chk1 and, for comparison, Nek2 (a centrosomal protein) and Mcm7 (a nuclear protein). For whole cell lysates, actin was included as a loading control. (E) Western blot analysis of PCNT, MCPH1, and Chk1 in whole cell lysates from control, MCPH1 427insA , and PCNT 3109G>T LBCs. Actin was included as a loading control. Arrowheads point to centrosomes, which are shown enlarged in insets.

Article Snippet: Mouse monoclonal antibodies to Cdk1 and GFP (B-2), rabbit antiserum to actin (I-19), Chk1 (FL-476), and HA (Y11) as well as goat and donkey antisera to HRP were obtained from Santa Cruz Biotechnology, Inc.; a mouse monoclonal antibody to Chk1 (DCS-310) and a rabbit antiserum to γ-tubulin were obtained from Sigma-Aldrich; a mouse monoclonal antibody to γ-tubulin (TU-30) was obtained from Exbio; a mouse monoclonal antibody to Nek2 was obtained from BD; a goat antiserum to MCPH1 was obtained from R&D Systems; rabbit antiserum to PCNT, P-Y15-Cdk1, and P-S10–histone H3 were obtained from Abcam, EMD, and Millipore, respectively.

Techniques: Transfection, Luciferase, Control, Confocal Microscopy, Standard Deviation, Two Tailed Test, Construct, Fluorescence, Isolation, Western Blot, Comparison

Journal: The Journal of Cell Biology

Article Title: Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1

doi: 10.1083/jcb.200810159

Figure Lengend Snippet: MCPH1 interacts with and targets Chk1 to the centrosome. (A) Control, MCPH1 427insA , and PCNT 3109G>T LBCs as well as U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA were costained with mouse anti-MCPH1 (red) and rabbit anti–γ-tubulin (green) antibodies and analyzed by confocal microscopy. (B) Reduced levels of PCNT in isolated centrosome preparations from MCPH1 427insA LBCs. Immunoblots were performed on three sucrose gradient fractions of centrosome preparations (left) and whole cell lysates (right) as an input control from normal, MCPH1 427insA , and PCNT 3109G>T LBCs using antibodies against PCNT, MCPH1, and, for comparison, Nek2 as a loading control and Mcm7 to exclude nuclear contamination. For whole cell lysates, actin was included as a loading control. (C) Transiently expressed GFP-MCPH1 and Chk1 interact with each other in vivo. Chk1 was immunoprecipitated from U2OS cells 24 h after transient transfection with GFP-MCPH1. Reciprocally, GFP-MCPH1 was immunoprecipitated using an anti-GFP antibody. Immunoprecipitation with an anti-HA antibody served as a negative control. Input represents 10% of the amount used for immunoprecipitation. White lines indicate that intervening lanes have been spliced out. (D) Depletion of Chk1 does not impact on centrosomal MCPH1 levels. U2OS cells transfected with either luciferase- or Chk1-specific siRNA were costained with mouse anti-MCPH1 (red) and rabbit anti–γ-tubulin (green) antibodies (top) or mouse anti-Chk1 (red) and rabbit anti–γ-tubulin (green) antibodies (bottom) and analyzed by confocal microscopy. Arrowheads point to centrosomes, which are shown enlarged in insets. Bars, 10 µm.

Article Snippet: Mouse monoclonal antibodies to Cdk1 and GFP (B-2), rabbit antiserum to actin (I-19), Chk1 (FL-476), and HA (Y11) as well as goat and donkey antisera to HRP were obtained from Santa Cruz Biotechnology, Inc.; a mouse monoclonal antibody to Chk1 (DCS-310) and a rabbit antiserum to γ-tubulin were obtained from Sigma-Aldrich; a mouse monoclonal antibody to γ-tubulin (TU-30) was obtained from Exbio; a mouse monoclonal antibody to Nek2 was obtained from BD; a goat antiserum to MCPH1 was obtained from R&D Systems; rabbit antiserum to PCNT, P-Y15-Cdk1, and P-S10–histone H3 were obtained from Abcam, EMD, and Millipore, respectively.

Techniques: Control, Transfection, Luciferase, Confocal Microscopy, Isolation, Western Blot, Comparison, In Vivo, Immunoprecipitation, Negative Control

Journal: The Journal of Cell Biology

Article Title: Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1

doi: 10.1083/jcb.200810159

Figure Lengend Snippet: MCPH1 recruits Chk1 to the centrosome via PCNT. (A) Centrosomal PCNT levels are reduced in MCPH1 427insA and PCNT 3109G>T LBCs relative to control lymphoblasts. Normal, MCPH1 427insA , and PCNT 3109G>T LBCs as well as U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) antibodies and analyzed by confocal microscopy. (B) Quantification of pixel intensity profiles constructed from optically sectioned ( z axis) fluorescence images of normal cells. Error bars represent standard deviations from the analysis of 100 cells. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 7.2 × 10 −6 (MCPH1 427insA ). (C) Control and Chk1 −/− chicken DT40 cells as well as U2OS cells transfected with luciferase- or Chk1-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) and analyzed by confocal microscopy. (D) Endogenous PCNT and Chk1 interact with each other in vivo. Endogenous Chk1 was detected in immunoprecipitates using an anti-PCNT antibody in both control and MCPH1 427insA LBCs. Reciprocally, endogenous PCNT was detectable after immunoprecipitation of Chk1 in both cell lines as well. Immunoprecipitation with an anti-HA antibody served as a negative control. Input represents 10% of the amount used for immunoprecipitation. White lines indicate that intervening lanes have been spliced out. (E) Cofractionation of PCNT, MCPH1, and Chk1 in U2OS whole cell lysates. Lysates were prepared from U2OS cells transiently transfected with GFP-MCPH1 24 h before lysis and size fractionated by fast protein liquid chromatography using a Superose 6 column. Proteins from consecutive fractions were analyzed by Western blotting using antibodies to PCNT, GFP, and Chk1. The size of marker proteins is shown on top. Numbers in red indicate the fractions that contain all three proteins (GFP-MCPH1, PCNT, and Chk1). Arrowheads point to centrosomes, which are shown enlarged in insets. Bars, 10 µm.

Article Snippet: Mouse monoclonal antibodies to Cdk1 and GFP (B-2), rabbit antiserum to actin (I-19), Chk1 (FL-476), and HA (Y11) as well as goat and donkey antisera to HRP were obtained from Santa Cruz Biotechnology, Inc.; a mouse monoclonal antibody to Chk1 (DCS-310) and a rabbit antiserum to γ-tubulin were obtained from Sigma-Aldrich; a mouse monoclonal antibody to γ-tubulin (TU-30) was obtained from Exbio; a mouse monoclonal antibody to Nek2 was obtained from BD; a goat antiserum to MCPH1 was obtained from R&D Systems; rabbit antiserum to PCNT, P-Y15-Cdk1, and P-S10–histone H3 were obtained from Abcam, EMD, and Millipore, respectively.

Techniques: Control, Transfection, Luciferase, Confocal Microscopy, Construct, Fluorescence, Two Tailed Test, In Vivo, Immunoprecipitation, Negative Control, Lysis, Fast Protein Liquid Chromatography, Western Blot, Marker

Journal: The Journal of Cell Biology

Article Title: Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1

doi: 10.1083/jcb.200810159

Figure Lengend Snippet: Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B and Cdk1. (A, C, and D) Normal, MCPH1 427insA , and PCNT 3109G>T LBCs were synchronized in G 1 /early S phase by a mimosine block, released for 8 h to reach G 2 phase, and subsequently costained with rabbit anti–P-S230-Cdc25B (green) and mouse anti–γ-tubulin (red) antibodies (A), rabbit anti–P-Y15-Cdk1 (green) and mouse anti–γ-tubulin (red) antibodies (C), or mouse anti-Cdk1 (red) and rabbit anti–γ-tubulin (green) antibodies (D) and analyzed by confocal microscopy. (B) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B. The mean percentages of cells with centrosomal colocalization of γ-tubulin and P-S230-Cdc25B are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 0.0006 (MCPH1 427insA ); **, P = 0.006 (PCNT 3109G>T ). (E) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdk1. The mean percentages of cells with centrosomal colocalization of γ-tubulin and total Cdk1 (light gray bars) or P-Y15-Cdk1 (dark gray bars) are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: *, P = 0.013 (MCPH1 427insA ); P = 0.019 (PCNT 3109G>T ). (F) Reduced levels of P-S230-Cdc25B and P-Y15-Cdk1 in isolated centrosome preparations from MCPH1 427insA and PCNT 3109G>T LBCs. Immunoblots were performed on three sucrose centrifugation fractions of centrosome preparations (left) and whole cell lysates (right) as an input control from control (LBC), MCPH1 427insA , and PCNT 3109G>T LBCs using antibodies against P-S230-Cdc25B, P-Y15-Cdk1, Cdk1, and, for comparison, Nek2 as a loading control and Mcm7 to exclude nuclear contamination. For whole cell lysates, antibodies to Cdc25B, Cdk1, and actin were included. Arrowheads point to centrosomes, which are shown enlarged in insets. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.

Article Snippet: Mouse monoclonal antibodies to Cdk1 and GFP (B-2), rabbit antiserum to actin (I-19), Chk1 (FL-476), and HA (Y11) as well as goat and donkey antisera to HRP were obtained from Santa Cruz Biotechnology, Inc.; a mouse monoclonal antibody to Chk1 (DCS-310) and a rabbit antiserum to γ-tubulin were obtained from Sigma-Aldrich; a mouse monoclonal antibody to γ-tubulin (TU-30) was obtained from Exbio; a mouse monoclonal antibody to Nek2 was obtained from BD; a goat antiserum to MCPH1 was obtained from R&D Systems; rabbit antiserum to PCNT, P-Y15-Cdk1, and P-S10–histone H3 were obtained from Abcam, EMD, and Millipore, respectively.

Techniques: Activation Assay, Blocking Assay, Confocal Microscopy, Control, Two Tailed Test, Isolation, Western Blot, Centrifugation, Comparison, Standard Deviation

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 2 | PEDV replication inhibit expression of SLA-DR in BM-DCs (A) BM-DCs were infected by Vero cell-adapted PEDV strain KB2013-p120 at 1MOI for 24, 48 and 72 hours then harvested for qPCR analysis for mRNA level of SLA-DRa and SLA-DRb. Transcript of tubulin were analyzed from the same sample to normalize total RNA input. Error bars represent variation from at least three independent experiments. Significant differences between indicated groups was marked by **P < 0.01; ***P < 0.001. (B) BM-DCs were infected by PEDV-KB2013-p120 strain at 1MOI for 24, 48 and 72 hours then harvested for western blot to evaluate SLA-DRa, SLA-DRb and PEDV-N protein level using corresponding antibodies. Normal BM-DCs cells without PEDV infection were included as control. Tubulin was probed from the same sample to normalize the total protein load. (C) BM-DCs were infected by PEDV-KB2013-p120 strain 1MOI for 24, 48 and 72 hours then stained with anti-SLA-DR antibody followed by visualization of APC labeled goat anti-mouse IgG. Then the cells were subjected to flow cytometry analysis for evaluating cell surface expression of SLA-DR. BM-DCs without PEDV infection stained with normal mouse IgG as primary antibody were included as primary antibody isotype control. Error bars represent variation of quantification of FACS data from at least three independent experiments. Significant differences between indicated groups was marked by **P < 0.01; or ns, non significant.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Expressing, Infection, Western Blot, Control, Staining, Labeling, Cytometry

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 3 | Susceptibility of BM-DCs for PEDV is not strain-specific. (A) BM-DCs were infected by PEDV-KB2013-p120 strain at 0.5, 1 and 2MOI for 48 hours then harvested for western blot to evaluate SLA-DRa, SLA-DRb and PEDV-N protein. Normal BM-DCs cells without PEDV infection were included as control. Tubulin was probed from the same sample to normalize the total protein load. (B) BM-DCs were infected by Vero cell-adapted PEDV strain KB2013-p120, virulent PEDV strain CH/hubei/2016 (GenBank accession number: KY496315.1), and a field PEDV isolate SXYL-21 for 48 hours. Next, cells were harvested for western blot using mice serum raised against recombinant PEDV-N protein and anti-SLA-DRa Mab. Normal BM-DCs cells without PEDV infection were included as control. Tubulin was probed from the same sample to normalize the total protein load. (C) BM-DCs were infected by PEDV-KB2013-p120 strain at 1 MOI for 24 hours then 10mg LPS was added to the PEDV infected BM-DCs to stimulate the cells for another 24 hours. Next, all cells were harvested for western blot to evaluate SLA-DRa, SLA-DRb and PEDV-N protein. PEDV infected BM-DCs without LPS stimulation and normal BM-DCs cells were included as controls. Tubulin was probed from the same sample to normalize the total protein load.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Infection, Western Blot, Control, Recombinant

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 5 | PAMs are susceptible for PEDV. (A) PAMs were infected by Vero cell-adapted PEDV strain KB2013-p120 for 24, 48 and 72 hours. Next, cells were harvested for western blot using mice serum raised against recombinant PEDV-N protein and anti-SLA-DRa Mab. Normal PAMs cells without PEDV infection were included as control. Tubulin was probed from the same sample to normalize the total protein load. (B) PAMs were infected by PEDV strain KB2013-p120 for 24, 48 and 72 hours. Next, cells were harvested by TRizol for qPCR analysis of SLA-DRa and b mRNA level. Normal PAMs cells without PEDV infection were included as control. Error bars represent variation from at least three independent experiments. Significant differences between indicated groups was marked by *P < 0.05; **P < 0.01. or ns, non significant.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Infection, Western Blot, Recombinant, Control

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 6 | Inhibition of SLA-DR expression in BM-DCs did not require PEDV replication. (A) BM-DCs were inoculated with either live PEDV-KB2013-p120 strain or UV-inactivated PEDV-KB2013-p120 strain for 24 hours. Next the cells were fixed and stained with mice serum raised against recombinant PEDV-N protein and visualized by secondary antibody. (B) BM-DCs were inoculated with either live PEDV-KB2013-p120 strain (PEDV) or UV-inactivated PEDV-KB2013-p120 strain (UV- PEDV) for 24 hours. Next, cell culture supernatants were harvested and titrated in Vero cells. Error bars represent variation from at least three independent experiments. (C) BM-DCs were inoculated with either live PEDV-KB2013-p120 strain (PEDV) or UV-inactivated PEDV-KB2013-p120 strain (UV-PEDV) for 24 hours. Next, cells were harvested for qPCR evaluation of mRNA level of SLA-DR a and SLA-DR b. All experiment was repeated at least for three times. Significant differences between indicated groups was marked by *P < 0.05. (D) BM-DCs were inoculated with either live PEDV-KB2013-p120 strain (PEDV) or UV-inactivated PEDV-KB2013-p120 strain (UV-PEDV) for 48 hours. Next, cells were harvested for western blot using mice serum raised against recombinant PEDV-N protein, anti- SLA-DRa Mab and mice serum raised against recombinant SLA-DRb protein. Normal BM-DCs cells were included as control. Tubulin was probed from the same sample to normalize the total protein load. ns, non significant.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Inhibition, Expressing, Staining, Recombinant, Cell Culture, Western Blot, Control

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 8 | Ubiquitin-proteasome pathway did not involve in inhibition of SLA-DR expression. (A) BM-DCs were infected by PEDV-KB2013-p120 strain at 1MOI for 24, then followed by treatment of either DMSO or MG132 for another 24 hours. Then cells were harvested for western blot evaluating of SLA-DRa, SLA-DRb and PEDV-N protein. Normal BM-DCs cells without PEDV infection but treated with the same dose of DMSO or MG132 were included as controls. Tubulin was probed from the same sample to normalize the total protein load. (B) BM-DCs were infected by PEDV-KB2013-p120 strain at 1MOI for 24 hours. Next, cells were harvested for western blot evaluating of universal ubiquitination level and PEDV-N protein. Normal BM-DCs cells were included as control. Tubulin was probed from the same sample to normalize the total protein load. (C) BM-DCs were infected by PEDV-KB2013-p120 strain at 1MOI for 24 hours. Next, cells were lyzed using RIPA buffer supplemented with protease inhibitor cocktail and NEM for SLA-DR enrichment using anti-SLA-DR Mab-MY533. Enriched samples were subjected to western blot using ubiquitin antibody and anti-SLA-DRa chain antibody. (D) HEK-293T-SLA-DRa/b stable cells were transfected with plasmids encoding S2, M, N, E and ORF3 for 48 hours. Next, cells were harvested for western blot to evaluate expression of SLA-DRa and b. Cells transfected with empty vector were included as control. Tubulin was probed from the same sample to normalize the total protein load.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Ubiquitin Proteomics, Inhibition, Expressing, Infection, Western Blot, Control, Protease Inhibitor, Transfection, Plasmid Preparation