EMBO/sd-character-level-ner huggingface dataset:
Figure 4A Box diagram of various fragments of PIF4 B, C Yeast two-hybrid assays showing the direct interaction between PIF4 and KIN10. Yeast clones were grown on the synthetic dropout medium with histidine (+HIS) or without histidine (-HIS) plus 25 mM of 3-amino-1,2,4-triazole (3-AT). D Co-IP assays showing the interaction between PIF4 and KIN10 in vivo. Protein extracts from protoplasts expressing PIF4-Myc and KIN10-GFP (or GFP) were immunoprecipitated with anti-GFP antibody and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. E GRIK1-activated KIN10 phosphorylates PIF4. In vitro phosphorylation assays were performed at 30 °C for 30 min using 1 μg of purified recombinant proteins (KIN10, GRIK1, or PIF4). 32P-labeled proteins were visualized by autoradiography. CBB, coomassie brilliant blue-stained gel. F T6P inhibits PIF4 phosphorylation by KIN10. This assay was performed with 1 μg of each protein at 30 °C for 30 min in the presence of 1 mM T6P or sucrose (Suc).
ACE2-Ig potently neutralizes viruses pseudotyped with S glycoproteins from the SARS CoV and 2019 nCoV.Figure 2.ACE2-Ig potently neutralizes viruses pseudotyped with S glycoproteins from the SARS CoV and 2019 nCoV.HIVs pseudotyped with the S glycoprotein from CoVs were incubated with different fusion proteins for 1 h before infection. Luciferase activities in target cells were measured, and the percent neutralization was calculated.
Fig.Figure 1 A-C. Positioning of Upf1, Upf2 and Upf3 in the abundance distribution of all yeast proteins, based on the data compiled by Ho et al., 2018 (A) The distribution of abundance for the proteins identified by mass spectrometry in input (B) show the expected bias for proteins present in high copy numbers The distribution of abundance for the proteins identified by mass spectrometry i purified samples (C), show the expected bias for proteins present in high copy numbers. Light grey colour highlights the positions of proteins that were only identified in the Upf1-TAP samples (6 replicates) and not in the total extract (3 replicates)  D. Correlation of LTOP2-based protein level estimates in a total yeast protein extract with published protein abundance data (Ho et al, 2018)  E. Distribution of the intensity for the proteins quantified in association with Upf1 (LTOP2 score, log2 scale). Positioning of Upf1 is highlighted as a white rectangle while Upf2 and Upf3 are highlighted in red. The dark grey line indicates the region of intensities corresponding to ribosomal proteins F. Distribution of the protein enrichment values for Upf1 associated proteins based on LTOP2 scores and known protein abundance in a total yeast extract. Upf1, Upf2 and Upf3 positions are also highlighted as white rectangles G. A combination of the data presented in (E) and (F) as a scatter plot, to see both the amount and enrichment of proteins in Upf1-TAP purified samples. The horizontal axis represents the LTOP2 score, with the vertical axis showing enrichment over input values. Both axes use log2 transformed values
a) RT–PCR analysis of LC3, IL-8, CHOP and GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM).f1(a) RT-PCR analysis of LC3, IL-8, CHOP and GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM). ( (b) Immunoblot analysis of ER stress- and autophagy-related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment. Samples are run in triplicate. (c) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. (d) IL-8 enzyme-linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. Error bars in all figures represent s.d. of three biological replicates.
TrackPal quantifies human cortical interneuron migration A.Figure 4A. Schematic overview of setup for live-imaging analysis of organoid fusions. Organoids at day 60-80 were sectioned with a vibratome to obtain 400µm thick sections. Dorsal regions of sections were then imaged every 20 minutes for three and a half days. B. Illustration of saltatory tangential migration performed by an interneuron. Nucleus is marked with a red asterisk. All images taken on a spinning disc confocal microscope at 10x magnification. C. Overview of different parameters calculated by TrackPal, which are used for track analysis of a track with straight-line motion. Global parameters provide initial assessment, after which the track is analyzed by either detailed global analysis parameters or segmented into tracklets. For global analysis, the confinement ratio provides information on the track straightness and the gyration tensor is used to analyze the area covered by a cell. For track segmentation, either spatiotemporal density-based clustering (DBSCAN) or the Ramer-Douglas-Peucker (RDP) simplification algorithm is used. Subsequently, detailed analysis is performed on the tracklets. Green and red dots mark the beginning and end of track. Yellow dots are nodes between different segments of the track in the RDP algorithm. Movement period of the cell is marked as "Moving". D. Overview of different parameters calculated by TrackPal, which are used for track analysis of a track with multiple directional changes and pausing periods. Parameters are analyzed as mentioned in 4C. Green and red dots signal the beginning and end of track. Yellow dots are nodes between different segments of the track in the RDP algorithm. Note, that the movement period (green) is separated by multiple phases of dwelling (red), that are marked as "Dwell" and "Moving" respectively.
The type II (Pru) Toxoplasma strain is more susceptible to IFNγ-mediated growth inhibition in primary human foreskin fibroblasts (HFFs) than the type I RH strain due to presence of the polymorphic effector GRA15.Figure 1A) HFFs were pre-stimulated with IFNγ (10U/mL) for 24 h. Plaque assays were performed for each strain and each condition. Plaque number and area loss were calculated 4 days p.i. for RH and 6 days p.i. for Pru. Assays were performed with RH (n=4), Pru (n=4). B) Relative parasite growth was measured 24 h p.i. in IFNγ-stimulated and unstimulated HFFs by luciferase assay. Growth of each strain in IFNγ-stimulated HFFs is expressed relative to growth in unstimulated HFFs. Experiments were performed with RH (n=4), Pru (n=4). C) Number of PVs was calculated using HFFs grown on coverslips and stimulated with 10 U/mL IFNγ for 24 h. Following stimulation, HFFs were infected with either RH (MOI=1) or Pru (MOI=3) for another 24 h. Coverslips were fixed and stained with GRA7 for parasite PVM and Hoechst 33258 for nuclei. The number of PVs in 5-6 fields of the coverslips were counted for each condition and normalized with the number of host cells in each field. Experiments were performed with RH (n=3), Pru (n=3). Representative images of percentage infected cells for each strain and condition are provided. Scale bar is 100 µm. D) Parasites per vacuole were determined 24 h p.i. with similar conditions and staining as described in (C) except MOIs used were 0.5 and 1 for RH (n=3) and Pru (n=3), respectively. E) Plaque assays were performed similarly as described for (a) with Pru WT (n=20 for plaque number and n=7 for plaque area), PruΔgra15 (n=20 for plaque number and n=7 for plaque area), PruΔgra15+GRA15 complemented (n=12 for plaque number and n=7 for plaque area). F) Plaque assays were performed similarly as described for (A) with RH (n=5 for plaque number and n=3 for plaque area) and RH+GRA15II (n=5 for plaque number and n=3 for plaque area). G) Relative parasite growth was measured as described in (B) with Pru WT (n=12), PruΔgra15 (n=12), PruΔgra15+GRA15 complemented (n=8). H) Number of PVs was calculated as described in (C) with Pru WT (n=3), PruΔgra15 (n=3), RH (n=3) and RH+GRA15II (n=3) and normalized with the number of host cells in each field.
Figure 1A Genomic organization of the human RBM15 locus. Exons are represented by black boxes. Arrows indicate transcriptional direction. 5'UTR and 3'UTR are represented by open boxes. B Expression levels of RBM15 and AS-RBM15 in different blood lineages by meta-analysis of RNA-seq data [38]: HSC (hematopoietic stem cells), MPP (multipotent progenitor cells), CMP (common myeloid progenitor cells), CLP (common lymphoid progenitor cells), GMP (Granulocyte-macrophage progenitor cells), MEP (megakaryocyte- erythrocyte progenitor cells), MK (megakaryocyte), and EB (erythroid body). C Relative expression levels of AS-RBM15 (left) and RBM15 (right) in MEG-01 cells treated with PMA were measured by real-time PCR assays (n=4, mean ± SD). SD: standard deviation. D Relative expression levels of AS-RBM15 and RBM15 in CD34+ cells grown in pro-MK medium were measured by real-time PCR. GAPDH was used as an internal control (n=3, mean ± SD). Also see Fig EV 1.
signalling in TAM affects the immune reactivity of T cells.Figure 5(A) Schematic of the analysis of bulk GL261 RNAseq data from Cx3cr1-Rheb1Δ/Δ (n=3) and Rheb1fl/fl (n=3) mice. ssGSEA enrichment scores for signalling pathway signatures and CIBERSORT cell fractions were calculated for each sample. Pearson correlation score was then calculated comparing signalling pathway enrichment scores and CIBERSORT cell fractions. Finally, a differential correlation analysis was run between the Cx3cr1-Rheb1Δ/Δ (n=3) and Rheb1fl/fl (n=3) samples. (B) Heatmap of the differential correlation analysis between CIBERSORT cell fractions and signatures enrichment scores (ssGSEA) for lymphocytes in the Cx3cr1-Rheb1Δ/Δ (n=3) vs Rheb1fl/fl (n=3) GL261 tumours. (C,D) CD4 Th cells (C) and CD8 CTL cells (D) expression of Ki67, perforin, granzyme b, and IFNγ, in Cx3cr1-Rheb1fl/fl (n=5) and Rheb1Δ/Δ (n=5) GL261 tumours. Representative FACS plots show control samples (tumour-derived cells stimulated in culture in the absence of protein inhibitor cocktail - in orange) compared to stimulated cells derived from Rheb1fl/fl (fl/fl, red) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, blue) GL261 tumours (cells stimulated in culture with protein inhibitor cocktail) (mean ±SEM; Two-Way ANOVA Tukey test). (E,F) Expression of CD44 (E) and CD62L (F) by CD4 Th and CD8 CTL in Cx3cr1-Rheb1fl/fl (n=5; fl/fl, red) and Rheb1Δ/Δ (n=5; Δ/Δ, blue) GL261 tumours with representative FACS plots show control samples (mean ±SEM; Two-Way ANOVA Tukey test).
Figure 4 Schematic illustrating the treatment of PK1 cells with ERdj3-conditioned media. HEK293T-Rex (Control) and HEK293T-Rex cells stably overexpressing tet-inducible ERdj3WT on poly-D-lysine coated plates were treated with dox (1 g/mL) overnight to induce ERdj3WT expression. The media was replaced with fresh OptiMEM I + 5% FBS, which was conditioned for 30 h, sterile-filtered, concentrated 5 X against a 3 kD MWCO membrane, and diluted 1:1.5 with fresh OptiMEM I + 5% FBS. PK1 cells were plated in OptiMEM I + 5% FBS, and after 24 h were treated with conditioned media and TPrP (600 ng/mL) for 3 d.   Representative images under 136X magnification of PK1 cells incubated with control conditioned media collected from HEK293T-Rex or conditioned media prepared from HEK293T-Rex cells overexpressing ERdj3WT in the presence or absence of TPrP (600 ng/mL), as described in Figure 4A. Arrows indicate representative cells containing vacuoles. Insets show 544X magnification of a typical region of the image. Quantification of vacuole formation from images as shown in Figure 4B. Equal area images comprising ~1200 cells per well were counted for each sample. Counting of cells and of cells with identifiable vacuoles was done using the Fiji image processing package (Schindelin et al, 2012). *** indicates a p-value < 0.001 (n = 3).
Restoring tissue oxygenation rescues radial glia differentiation after suppression of periventricular brain angiogenesis.Figure 6A, Scheme of the experimental approach. B-E, Staining for pimonodazole (PIMO, green) and Isolectin B4 (IB4,red) in Gpr124KO (B,C) and control (D,E) cortices after exposure of the pregnant dams to 21% O2 (B,D) or 80% O2 (C,E). Compared to the pimonidazole staining in panels B,C, the intensity of this staining is much weaker in panels D,E. For reasons of clarity, the pimonidazole staining in panels D,E was therefore enhanced, equally in both panels. F,G, Immunostaining for HIF-1 (red) and DAPI (blue) in Gpr124KOcortices after exposure of the pregnant dams to 21% O2 (F) or 80% O2 (G). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm H,I, Immunostaining for Tbr2 (red) and Ngn1 (green) in Gpr124KO cortices after exposure of the pregnant dams to 21% O2 (H) or 80% O2 (I). J-L Quantification of neurogenic (Ngn1+) RGs and BPs (J), newborn BPs (K) or expanding (Tbr2- Ngn1-) RGs (L) in Gpr124KO cortices after exposure of the pregnant dams to 21% O2 or 80% O2 (mean±SEM; N=4 for 21% O2 and N=5 for 80% O2; * p<0.05, ** p<0.01). M,N, Immunostaining for Tbr2 (red) and Ngn1 (green) in control cortices after exposure of the pregnant dams to 21% O2 (M) or 80% O2 (N). O-Q Quantification of neurogenic (Ngn1+) RGs and BPs (O), newborn BPs (P) or expanding (Tbr2-Ngn1-) RGs (Q) in control cortices after exposure of the pregnant dams to 21% O2 or 80% O2 (mean±SEM; N=5; ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm
a,b) Immunostaining using an α-ubiquitin antibody of (a) PCs and (b) the molecular layer of Miz1ΔPOZNes and age-matched control mice (dotted area: higher magnification).f5(a,b) Immunostaining using an α-ubiquitin antibody of (a) PCs and (b) the molecular layer of Miz1ΔPOZNes and age-matched control mice (dotted area: higher magnification). Arrowheads designate ubiquitin-positive structures. Scale bar, (a) 25 μm; (b) 50 μm. (c) Immunofluorescence staining using an α-p62 and an α-ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age-matched control mice. Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) and overviews of the cerebella clarifying the selected areas are shown. Scale bar, 25 μm. (a,b) Immunostaining using an α-ubiquitin antibody of (a) PCs and (b) the molecular layer of Miz1ΔPOZNes and age-matched control mice (dotted area: higher magnification). Arrowheads designate ubiquitin-positive structures. Scale bar, (a) 25 μm; (b) 50 μm. (c) Immunofluorescence staining using an α-p62 and an α-ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age-matched control mice. Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) and overviews of the cerebella clarifying the selected areas are shown. Scale bar, 25 μm. (d) Quantification of a, b and c. Error bars represent s.e.m. derived from the indicated numbers of animals. P-values were calculated using an unpaired two-tailed Student's t-test. (e) Immunoblots of Triton X-100 soluble ('TX100') and insoluble ('1% SDS') fractions of cerebella obtained from control and Miz1ΔPOZNes mice of the indicated ages. Blots were probed with antibodies recognizing either mono- or polyubiquitinated proteins. Staining of the membrane with amido black was used as loading control. (f) Transmission electron microscopy documenting the accumulation of large multilamellar bodies in PC bodies ('PC') and Purkinje dendrites ('D') of Miz1ΔPOZNes mice (N, nucleus). Such structures were not observed in age-matched control mice (as quantified in Supplementary Fig. S7b). Mean age of mice for all electron microscopy pictures is 13 monthsfor Miz1ΔPOZNes and 12.5 months for control mice. Morphologically similar structures accumulate in neurons of mice deficient in late steps of autophagy. Scale bars, 2, 0.2, 0.5 and 1 μm, respectively.
GAPDH selectively associates with mitochondria in cells expressing expanded polyglutamine repeats.Figure 1Representative Western blots showing GAPDH association with mitochondria-enriched fractions in different HD models. The levels of GAPDH were normalized to mitochondrial matrix protein, aconitase. n = 3. *P = 0.0005; **P = 0.019; #P = 0.042. Measurement of GAPDH activity in the mitochondria-enriched fraction isolated from PC12 cells. n = 3. *P = 0.0005. Western blots showing oxidized GAPDH in the total lysates of PC12 cells and isolated mitochondria. The levels of oxidized GAPDH were normalized to beta-actin (total lysate) and aconitase (mitochondrial fraction), respectively. n = 2. *P = 0.023; **P = 0.039. Western blot showing co-immunoprecipitation of GAPDH from the total lysates of PC12 cells in RIPA buffer without detergent (Triton X-100). Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial (VDAC and Tom20), endoplasmic reticulum (calnexin), and lysosomal markers (LAMP1). ctr: control. n = 2.
A proposed model of HOIP-site specific ubiquitination in the regulation of the TNF signaling cascades.Figure 6A working model describing a role of site-specific ubiquitination of HOIP in the regulation of the TNF-induced NF-κB and apoptosis pathways based on this study. Schematics of simplified TNF signaling in wild type cells (WT, left panel) and HOIP-K784R cells (right panel) are shown. Upon TNF-binding to TNF receptor 1, complex I, which includes RIPK1 and LUBAC components (HOIP, HOIL-1L and SHARPIN) is formed. In this signaling cascade, LUBAC linearly ubiquitinates NEMO and RIPK1. Site specific ubiquitination of HOIP at K784 plays a role in inducing TNF-dependent NF-κB target genes and in inhibition of apoptosis by inhibiting RIPK1 recruitment into TNF receptor complex II. Linkage types of ubiquitin chains conjugated on HOIP-K784 remains open.
NPC components aid the enrichment of HXT2 mRNA in the bud upon refeeding.Figure 6A Deletion of NUP2 and MLP1/2 reduced HXT2 mRNA enrichment in the bud. FISH under glucose shift conditions with strains in which different NPC components were deleted. B Quantification of (A). C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift. FISH-IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84-HA. White arrows point to HXT2 mRNA signals on the nuclear envelope. D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift. Stills of time lapse movies. HXT2 mRNA was visualized using the MS2-GFP system. Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus. Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus. Cyan: Nup84-mCherry; white arrowhead: HXT2 mRNA-MS2 + MCP-GFP.
Figure 1 A. Structure of budding yeast Rif1 and C-terminally truncated mutant Rif1-∆C594. RBM: Rap1-binding motif; CTD: C-terminal domain  B. Rif1-∆C594 retains function to control DNA replication. Growth of a cdc7-1 rif1-∆C594 mutant was compared with growth of cdc7-1 RIF1 and cdc7-1 rif1∆ strains at 23ºC (permissive temperature for cdc7-1 allele), 26ºC (mild restrictive temperature) and 30ºC (strict restrictive temperature)
Autophagic pathway progression promotes secretion of inflammasome substrates.Figure 2(A, B) LPS‐pretreated BMMs were treated with 20 μM nigericin (Nig) and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β (A) and IL‐18 (B) were measured. Data represent mean values±s.d. (n⩾3); *P0.05. (C) LPS‐pretreated BMMs were treated with 250 μg/ml of silica and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β were measured. Data represent mean values±s.d. (n⩾3); *P0.05. (D) Colocalization of cathepsin B with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), and cathepsin B (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were treated with LPS and then analysed for immunofluorescence. (E) Colocalization line tracing analysis from images in (D). (F) LPS‐pretreated BMMs were treated with 20 μM nigericin and cathepsin B inhibitor CA‐074 Me (10 μM), with (Starvation) or without (Full) autophagic induction, for 1 h and secreted IL‐1β was measured. Data represent mean values±s.d. (n⩾3); *P0.05. (G) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and release of cathepsin B was determined by immunoblotting. Figure source data can be found in Supplementary data.
The adjacent positions of the E239K mutation on β7 and the neck domain.Figure 5(A) Overall E239K structure. The WT structure is superimposed in the central panel in brown. The location of E239/K239 is shown in magenta, the nucleotide-binding domain (residues 98-102 and 189-216) is shown in blue, the microtubule-binding domain (residues 168-178, 249-309 and 335-353) is shown in purple, and the neck domain (residues 354-381) is shown in green. (B) Comparison of the WT (left) and E239K (right) around E239/K239. E239 and K239 are located on β7 and sandwiched between D363 of the neck-linker and β6 K228. The Cα-Cα distances of D363-E239/K239 and E239/K239-K228 were measured and are shown. NC, neck-coil; NL, neck-linker. The WT model is superimposed on the E239K model in brown. (C) Electrostatic charge distributions shown by the surface model. The regions of charge changes are indicated by dotted circles. Negative and positive charges are shown in red and blue, respectively.
Figure 6The model suggests the most likely interactions of CheY at the switch. Yellow ring, FliN. Green or red ring, FliMM in a conformation to which CheY can or cannot bind, respectively. Red background region is counterclockwise rotation. Yellow background region is frequent switching. Green shaded region is clockwise rotation. A, When the motor rotates counterclockwise, CheY most likely binds to FliMN because FliMN is a high-affinity site and because it is the most accessible site. Even though FliN is accessible to CheY, an interaction of CheY with FliN is not probable at physiological CheY concentrations, unless CheY is first trapped and potentiated by binding to FliMN. (Potentiated CheY is marked with an asterisk.) FliMM is probably sterically blocked for CheY binding or is in a conformation that does not favor CheY binding. B, FliMN-bound CheY has high probability of interacting with FliN. This interaction generates brief episodes of clockwise rotation, because the complex CheY-FliN is unstable and is likely to quickly dissociate. C, During the short episodes of clockwise rotation shown in B, FliMM either becomes accessible to CheY or is changed to a conformation that favors CheY binding. As a result, CheY (or, most likely, CheY~Ac) binds to FliMM. This binding stabilizes the clockwise conformation of FliMM and prevents the latter from resuming its counterclockwise conformation or becoming again sterically blocked for CheY binding. As long as CheY is bound to FliMM, CheY dissociation from FliN no longer affects clockwise rotation. CheY dissociation from FliMM when CheY is already dissociated from FliN would cause an immediate return to counterclockwise rotation.