Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Journal of Cell Science

Article Title: The PP2A B56 phosphatase promotes the association of Cdc20 with APC/C in mitosis

doi: 10.1242/jcs.201608

Figure Lengend Snippet: PP2AB56 promotes the timing of anaphase and mitotic exit. (A–C) PP2AB56 depletion delays anaphase onset. (A,B) RPE1 cells expressing RNAi-resistant B56α and/or H2B–mCherry were transfected with siRNA targeting B56α, B56β, B56γ, B56δ and B56ε (siB56α-ε) or control siRNA (siCtrl) and imaged live. (A) The duration of mitosis was measured for individual cells. Plotted is the fraction of cells that entered anaphase as a function of time in mitosis. (B) Representative micrographs. Numbers indicate time (min) relative to nuclear envelope breakdown (NEBD). The time to metaphase (blue bar) and time from metaphase to anaphase (green bar) is indicated. (C) RPE1 cells expressing H2B–mCherry were treated with siCtrl, siB56α-ε, or a mixture of siRNAs against B56α, B56γ, B56δ and B56ε (siB56) and imaged live. The cumulative anaphase onset is plotted. (D) Depletion of PP2AB56 delays mitotic exit. RPE1 cells were treated with siCtrl, siB56α-ε or mixtures in which an siRNA against a single B56 isoform had been omitted. Cells were treated with nocodazole for 2 h and then imaged live before and after reversine addition. The median time of mitotic exit (line) and exit time of individual cells (open circles) is plotted. Triangles indicate cells that remained in mitosis at the end of the imaging period. (E) siCtrl and siB56 mitotic HeLa lysates were probed by western blotting. Asterisk indicates non-specific band. (F,G) Cyclin B1 proteolysis is delayed in siB56 cells. (F) RPE1 CCNB1Venus/+ siCtrl or siB56 cells were treated as described in D and imaged live. Still images from differential interference contrast (DIC) and fluorescent imaging are shown. Numbers indicate time (min) relative to addition of reversine. (G) Plotted is the fluorescence intensity relative to reversine addition. Each line indicates a single cell and the last time point plotted is either mitotic exit or the experimental end-point (150 min). (H,I) siB56 cells have increased recruitment of Mad2 and BubR1 when Mps1 is inhibited. RPE1 siCtrl or siB56 cells were incubated with nocodazole and MG132, and treated with or without reversine before processing for immunofluorescence microscopy. (H) Maximum intensity projection images of representative cells used for quantification shown in I. (I) Quantification of kinetochore recruitment. Each circle represents the average kinetochore signal of one cell. Line indicates mean; a.u., arbitrary units; n.s., not significant (P>0.05), **P<0.005, ***P<0.0005, Student's two-tailed t-test. Results are representative of three independent experiments. n indicates number of cells analyzed from three experiments (A,C) or single experiment (D). Scale bars: 5 μm.

Article Snippet: Commercial antibodies used in this study include those against: α-tubulin (mouse DM1α–FITC conjugate; Sigma, F2168); β-actin (Sigma, A5316); APC1 (Bethyl Laboratories, A301-653A); APC3 (BD Biosciences, 610455, western blot only); APC7 (Bethyl Laboratories, A302-551A); APC8 (Bethyl Laboratories, A301-182A); BubR1 (BD Transduction Laboratories, 612503; immunofluorescence and western blot only); PP2A catalytic subunit (BD Transduction Laboratories, 610555); PP2A scaffold (Santa Cruz Biotechnology, sc-6112); Mad2 (Bethyl Laboratories, A300-301A); B56γ (Bethyl Laboratories, A303-814A); B56δ (Bethyl Laboratories, A301-098A); and human CREST antiserum (Immunovision, Springsdale, AR, HCT-0100; 1:5000).

Techniques: Expressing, Transfection, Imaging, Western Blot, Fluorescence, Incubation, Immunofluorescence, Microscopy, Two Tailed Test

Journal: Journal of Cell Science

Article Title: The PP2A B56 phosphatase promotes the association of Cdc20 with APC/C in mitosis

doi: 10.1242/jcs.201608

Figure Lengend Snippet: PP2AB56-dependent stimulation of APC/CCdc20 assembly does not require direct binding between B56 and BubR1. (A) BubR1 association with APC/C is reduced in siB56 cells. Mitotic siB56 or siCtrl HeLa cells were used for BubR1 (lanes 4,5) or control (lane 3) IgG IPs and analyzed by western blotting. (B) Quantification of three replicate experiments as in (A). (C–H) Deletion of the KARD in BubR1 does not alter APC/C–Cdc20 association. (C) Schematic for BubR1 depletion and rescue in HeLa cells. (D) BubR1-rescue lysates were analyzed alongside lysates from untreated cells by quantitative western blotting. Endogenous BubR1 signals were normalized to the actin signal, and the value in the untreated sample was set to 1. Solid line indicates intervening lanes have been cropped. (E–H) Lysates (lanes 1,2) and IPs (lanes 3-5) of GFP (E) or Cdc20 (G) were analyzed by western blotting. The experiments in E,G were performed three times and the quantifications are shown in F and H, respectively. (I,J) Comparison of mitotic exit delay in siB56 cells versus BubR1-rescue cells. siCtrl and siB56 cells (I) or BubR1-rescue cells (J) were incubated in nocodazole and reversine and imaged live. Plotted is the fraction of cells that exited mitosis as a function of time after mitotic entry. n, total number of cells analyzed from three independent experiments. Bars are mean±s.d. n.s., not significant (P>0.05); *P<0.05, **P<0.005, ***P<0.0005, Student's two-tailed t-test.

Article Snippet: Commercial antibodies used in this study include those against: α-tubulin (mouse DM1α–FITC conjugate; Sigma, F2168); β-actin (Sigma, A5316); APC1 (Bethyl Laboratories, A301-653A); APC3 (BD Biosciences, 610455, western blot only); APC7 (Bethyl Laboratories, A302-551A); APC8 (Bethyl Laboratories, A301-182A); BubR1 (BD Transduction Laboratories, 612503; immunofluorescence and western blot only); PP2A catalytic subunit (BD Transduction Laboratories, 610555); PP2A scaffold (Santa Cruz Biotechnology, sc-6112); Mad2 (Bethyl Laboratories, A300-301A); B56γ (Bethyl Laboratories, A303-814A); B56δ (Bethyl Laboratories, A301-098A); and human CREST antiserum (Immunovision, Springsdale, AR, HCT-0100; 1:5000).

Techniques: Binding Assay, Western Blot, Incubation, Two Tailed Test

Journal: Journal of Cell Science

Article Title: The PP2A B56 phosphatase promotes the association of Cdc20 with APC/C in mitosis

doi: 10.1242/jcs.201608

Figure Lengend Snippet: B56α interacts with the APC/C constitutively and independently of BubR1. (A,B) B56α co-precipitates with APC/C. (A) Mitotic (mit.) or asynchronous (asy.) HeLa cells were subjected to IP for B56α (lanes 4,5) or with control IgG (lane 3) and probed by western blotting for the indicated proteins. (B) Quantification of three replicate experiments as described in A. Values were normalized to those for the PP2A catalytic subunit (PP2A cat.). (C,D) Mitotic interaction of B56α and APC/C does not require the KARD of BubR1. BubR1 depletion and rescue was performed as described in Fig. 4C, and cells were subjected to IP for B56α (lanes 4,5) or with control IgG (lane 3) and probed by western blotting. (D) Quantification of three replicate experiments as described in C. (E,F) An LxxIxE motif mediates association of APC/C with B56α. Expression of YFP–B56α WT or an R222E mutant was induced by doxycycline in HeLa FLP-In lines, and mitotic cells were collected. (E) IPs for GFP (for YFP-tagged constructs; lanes 4,5) and control (lane 3) IPs were analyzed by western blotting. Asterisk indicates non-specific band. (F) Quantification of three replicate experiments described in E. Data are mean±s.d. n.s., not significant (P>0.05); *P<0.05, **P< 0.005, ***P<0.0005, ****P<0.00005, Student's two-tailed t-test.

Article Snippet: Commercial antibodies used in this study include those against: α-tubulin (mouse DM1α–FITC conjugate; Sigma, F2168); β-actin (Sigma, A5316); APC1 (Bethyl Laboratories, A301-653A); APC3 (BD Biosciences, 610455, western blot only); APC7 (Bethyl Laboratories, A302-551A); APC8 (Bethyl Laboratories, A301-182A); BubR1 (BD Transduction Laboratories, 612503; immunofluorescence and western blot only); PP2A catalytic subunit (BD Transduction Laboratories, 610555); PP2A scaffold (Santa Cruz Biotechnology, sc-6112); Mad2 (Bethyl Laboratories, A300-301A); B56γ (Bethyl Laboratories, A303-814A); B56δ (Bethyl Laboratories, A301-098A); and human CREST antiserum (Immunovision, Springsdale, AR, HCT-0100; 1:5000).

Techniques: Western Blot, Expressing, Mutagenesis, Construct, Two Tailed Test