EMBO/sd-character-level-ner huggingface dataset:
Figure 7Studies show that Opa1 interacts with MICOS complex, promoting closure of CJs. Thus, we tested whether Opa1 was required to maintain the difference in ΔΨm between cristae and IBM. Live-cell Airyscan images of MEF control (top row) vs. Opa1 KO (bottom row), co-stained with NAO and TMRE. Arrowheads indicate decreased intensity of TMRE FI at cristae compared to IBM. Note the more even distribution of TMRE staining, indicating the cristae and IBM are relatively equipotential. Scale bar = 500 nm. Quantification of G, showing Opa1-KO MEFs have significantly decreased ΔΨCr-IBM. N = 3 independent experiments.
Figure 3(A) Multiple sequence alignment of the Cyclin B2 and Cyclin B1 proteins from various species as indicated. The sequence alignment was developed by ClustalW2 software. (B) Flag immunoprecipitation assay. Flag-tagged Cyclin B2-WT, Cyclin B2-4A, or Cyclin B1 were expressed in 293T cells and purified with anti-Flag M2 beads. Subsequently, the beads were incubated with mitotic HeLa lysates for 2 h, and, following extensive washes, immunoprecipitatates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-Mad2 antibody. (C) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-Cyclin B2-WT or Flag-Cyclin B2-4A together with GFP (negative control) and GFP-CDK1. After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitation samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-GFP antibody and anti-Flag antibody. (D) Representative immunofluorescence images of HeLa cells transfected with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for ACA (red) and DNA (blue). (E) Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2-WT/4A treated as in D. Bars represent the mean kinetochore intensity (±SD) normalized to the values of GFP-Cyclin B2-WT. Each dot represents one cell (≥30 cells from three independent experiments).
Figure 3A The nickel-pull down assay using myc/HIS-tagged ENTREP along with HA-tagged ubiquitin. Arrows indicate HA-ubiquitin-incorporating ENTREP. Arrowhead indicates ENTREP without ubiquitination. Data shown are representative of three independent experiments. B The ubiquitination of ENTREP. The immunoprecipitated samples using anti-FLAG antibody under the denaturing condition were separated by the SDS-PAGE. The gel area corresponding to band #A, #B and #C of ENTREP-FLAG were applied to the ubiquitin-AQUA/PRM analysis. The absolute number of each ubiquitins of ENTREP #A, #B and #C were calculated by subtraction of those of the corresponding area of the control vector sample (Appendix Table S2 for raw data of the ubiquitin-AQUA/PRM analysis). Based on the ratios of each ubiquitins to the total ubiquitin of indicated gel area, which are showed as Circular graphs (ENTREP #A, #B and #C), the number of ENTREP-linked ubiquitin (Ub) is summerized as schema: #A is modified with one Ub, and about two thirds of #B molecules and half of #C molecules are modified with two and three of single Ubs (multi-monoubiquitin), respectively. The result was based on three biological replicates.
Figure 8(A and B) ΔΨm (A) and Acly levels (B) in HSCs derived from 5-FU treated mice in the late phase before and after a 1-day culture. (C) The frequencies of the CD150+CD48- and CD48+ fractions within the lineage-EPCR+ fraction after a 4-day culture of HSCs in the late phase with or without BMS303141 (BMS; 100 μM), SB204990 (SB; 100 μM), an alternative Acly inhibitor, or CTPI-2 (50 μM). (D and E) Total cell number (D) or CD48+ cell number (E) after the 4-day culture of HSCs in the late phase with or without BMS303141 (BMS), SB204990 (SB), or CTPI-2. Numbers within the graph represent the inhibitory effects of each treatment (fold decrease by an inhibitor treatment). (F) Global H3K27ac in HSCs in the late phase before or after a 1-day culture with or without BMS303141 (BMS), SB204990 (SB), or CTPI-2. (G) The frequencies of CD150+CD48- and CD48+ fractions within lineage-EPCR+ after HSCs in the late phase were cultured for 4 days with or without A-485 (1 μM), an inhibitor of histone acetyltransferase. (H and I) Total cell number (H) or CD48+ cell number (I) after a 4-day culture of HSCs in the late phase with or without A-485. Numbers within the graph represent the inhibitory effects of each treatment (fold decrease by an inhibitor treatment). (J) The frequencies of CD150+CD48- and CD150+CD48+ fractions within lineage-EPCR+ after a serial CTPI-2 treatment (50 mg/kg/shot/day) following the administration of 5-FU in mice transplanted with 15000 HSCs without irradiation.
Figure 6A,C. Immunoblot analysis for modified histone in anti-FLAG pulldown of UBR7-FL (A) or sNASP-FL (C) from non-nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors (hydroxyurea, aphidicolin) or transcription inhibitors (α-amanitin, triptolide). B,D. Plots represent either normalized immunoblot signals or cell counts prior to fractionation. Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation, with grey dotted line marking mean of No Treat condition. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiments were performed three times. Cells numbers were counted in two of the three experiments.
Figure 4(A) Surface representation of the GAPC1 tetramer (dimer of O-R-dimer) with colors indicating the different chains and respective sequence identity (O=O´and R=R´) and highlighting the conformations of Tyr-Asp (blue) docked to the identified pockets, labelled 1-3, as well as the catalytic site (Cys 149 and His 176 in red), and NAD+ (pink). (B) Predicted binding conformation of Tyr-Asp docked to GAPC1 without NAD+ (but shown for reference and taken from 4z0h, purple). (C) Zoom-in pocket 1 and 2 with overlaid Cp12-2 (taken from PDB-ID 3qv1, cartoon gray) with highlighted glutamic acid in sequence position 12 and C-terminal Tyr-Asp motif (both in stick representation), which sterically interfere with Tyr-Asp binding to pocket 1 and 2.
Figure 4(A) PPARγ target genes expression measured by qPCR (n=5-12 each group, two-way ANOVA, post hoc Holm-Sidak comparisons *p<0.05, **p<0.01). (B) Immunoblot analysis of PPARγ in nuclear fraction. On the right, quantification of PPARγ normalized to p84 is shown. Average of donor mice [19] and 4 experimental replicates per time-point, per group of recipient mice. (Two-way ANOVA, post hoc Holm-Sidak comparisons *p<0.05). (C) Immunoblot analysis of PPARγ in chromatin fraction. On the right, quantification of PPARγ normalized to the TBP is shown. Average of 4 experimental replicates, per time-point, per group (Two-way ANOVA, not significant difference). (D) Chromatin recruitment of PPARγ at PPAR binding site contained in Cidecand Pcxpromoters (n=3-5, per group, per ZT, Two-way ANOVA, post hoc Holm-Sidak comparisons *p<0.05). H3K4me3 abundance at Cidecand Pcxpromoters (n=4-7, per group, per ZT, unpaired, two-tailed Student t-test *p<0.05). (E) Pparγ and Cidec gene expression at ZT12 in the liver of fecal-transplanted animals treated with GW9662 or Vehicle (n=6-9, per group, Two-way ANOVA, post hoc Holm-Sidak comparisons *p<0.05). (F) % Epididymal fat mass/body weight of fecal-transplanted animals treated with GW9662 or Vehicle (n=6-9 per group, unpaired, two-tailed Student t-test *p<0.05).
Figure 7A. Detergent-solubilized yeast extracts from cells containing chromosomal Flag-tagged rbd2 or rbd2-G246R in rbd2Δ background were prepared, and proteins associated with Rbd2-Flag were immunopurified using anti-Flag magnetic beads. Equal amount of total (lanes 1-3) along with 25x bound fractions (lanes 4-6) were analyzed by western blot with anti-Cdc48 serum and anti-FlagIgG. B. Illustration outlining APEX2-catalyzed biotin-phenol labeling. An Rbd2-Flag-APEX fusion protein oxidizes biotin-phenol to biotin-phenoxyl radical that covalently labels binding partners. C. Biotin-phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods. WT cells or rbd2Δ cells carrying rbd2-Flag-APEX2 (rbd2-F-AX) or rbd2-G246R-Flag-APEX2 (G246R-F-AX) plasmid were lysed after biotin-labeling reaction, and proteins were denatured by heating the cells in lysis buffer containing 1%SDS. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates and 50x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Cdc48 serum, or anti-FlagIgG.
Figure 8(A) W1118 and TBPH-/- larvae were blotted using antibodies against phosphorylated p70S6K (T398), phosphorylated 4E-BP1 (T37/46) and tubulin antibodies. (B) Total RNA was prepared from W1118 and TBPH-/- larvae and subjected to RT-PCR analysis. Ribosomal protein L32 (RPL32) was used as loading control.(C) Total RNA was prepared from W1118 and TBPH-/- larvae and subjected to qRT-PCR analysis. The level of TBPH and raptor were quantified and normalized relative to RPL32. Data from three independent experiments represented as means ± S.E.M., **, p<0.01, one-way ANOVA. (D) The heads of W1118 and TBPH-/- larvae were cut and blotted using antibodies against phosphorylated p70S6K (T398), phosphorylated 4E-BP1 (T37/46) and tubulin antibodies. (E) The first, second and third instar larvae of W1118 and blotted-/- were blotted using antibodies against phosphorylated p70S6K (T398), phosphorylated 4E-BP1 (T37/46) and tubulin antibodies. (F-H) Similar experiments as in (A-C) were performed in W1118 and TBPH-/- adult flies. The data from three independent experiments indicated the means ± S.E.M., **, p<0.01, one-way ANOVA. W1118 and TBPH-/- larvae were blotted using antibodies against phosphorylated p70S6K (T398), phosphorylated 4E-BP1 (T37/46) and tubulin antibodies. (F-H) Similar experiments as in (A-C) were performed in W1118 and TBPH-/- adult flies. The data from three independent experiments indicated the means ± S.E.M., **, p<0.01, one-way ANOVA. Total RNA was prepared from W1118 and TBPH-/- larvae and subjected to RT-PCR analysis. Ribosomal protein L32 (RPL32) was used as loading control. Total RNA was prepared from W1118 and TBPH-/- larvae and subjected to qRT-PCR analysis. The level of TBPH and raptor were quantified and normalized relative to RPL32. Data from three independent experiments represented as means ± S.E.M., **, p<0.01, one-way ANOVA. (I) Similar experiments as in (C) were performed in W1118 and TBPH-/- adult flies. The mRNA levels of lysosomal and autophagic genes were quantified and normalized relative to GAPDH. The data from three independent experiments are presented as means ± S.E.M., ns, not significantly different; **, p<0.01, one-way ANOVA.Total RNA was prepared from W1118 and TBPH-/- larvae and subjected to qRT-PCR analysis. The level of TBPH and raptor were quantified and normalized relative to RPL32. Data from three independent experiments represented as means ± S.E.M., **, p<0.01, one-way ANOVA.
Figure 4(A) DIV 4 cortical neurons expressing p110 isoforms and GFP. Arrow marks the axon tip. (B) Quantification of axon length. n = 3 experiments, 45 neurons per group. (C) Quantification of dendrite length. n = 3 experiments, 45 neurons per group. (D) Quantification of the axon:dendrite length ratio. n = 3 experiments, 45 neurons per group. (E) DIV 14 cortical neurons expressing p110 isoforms and GFP, immunolabelled for pS6. (F) Sholl analysis of branches. n = 60 neurons per group. Data are shown as the mean +/- SEM. P values indicate significance measured by repeated measure ANOVA with Bonferonni's post-hoc test. (G) Quantification of the total neurite length. n = 3 experiments, 60 neurons per group. (H) Quantification of soma area. n = 3 experiments, 60 neurons per group. (I) Quantification of the pS6 immunofluorescence. n = 3 experiments, 60 neurons per group.
Figure 2(A) Comparison of observed rates of elongation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA). (B) Comparison of total peptide formation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA). (C) Elongation kinetics for the CGA-CGA inhibitory codon pair with the native arginine ICGtRNA (red) or the non-native arginine UCGtRNA (pink) and for the CGC-CGC optimal control pair with the native arginine ICGtRNA (green).
Figure.Figure 1A. Schematic view of the transgenic mice, ICSI and SCNT experiments. ♂ and ♀ indicated the male and female, respectively B. Representative immunofluorescence and live-cell images of dynamics MERVL::tdTomato and Gag expression during embryos preimplantation development (left). Quantification of tdTomato and Gag intensity (right). For the live-cell images, average intensity of tdTomato signal intensities relative to 2-cell stage embryos. For the immunofluorescence images, bar graphs showing the relative intensities of Gag/DAPI signal ratio. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 4. **P < 0.01, ***P < 0.001 by two-tailed Student"s  t-test. Scale bar, C. Western blot analysis MERVL::tdTomato levels in MII oocyte and embryos at the indicated stages (upper). GAPDH was used as a loading control. Numbers below the Western blots indicate band intensity (normalized to total GAPDH) measured by using ImageJ software. Quantification of Western blot results (bottom). MII, MII oocyte; M, morula; B, blastocyst. Error bars, s.d., n = 3. **P < 0.01, ***P < 0.001 by two-tailed Student"s  t D. Representative fluorescence image of 2-cell embryos derived from ICSI or SCNT. The SCNT embryos produced by transfer of MERVL::tdTomato cumulus cell into WT enucleated oocytes. ICSI embryos produced by MERVL::tdTomato sperm and MII oocytes from the littermates of transgenic mice. Scale bar, 50 μm E. The summary of tdTomato+ 2-cell embryos derived from ICSI or SCNT. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 3. ***P < 0.001 by two-tailed Student"s  t F. The endogenous MERVL was up-regulated significantly in ICSI embryos compared with SCNT embryos at 2-cell stages, as determined by RT-qPCR. Error bars, s.e.m., n ≥ 3 G. RT-qPCR data for select ZGA genes activated following MERVL::tdTomato expression in mouse 2-cell embryos derived from ICSI or SCNT. Results were normalized based on the geometric mean of the expression levels of two reference genes (Ywhaz and Gapdh). Error bars, s.e.m., n = 3. ***P < 0.001 by two-tailed Student"s  t H. Representative images of tdTomato+ and tdTomato- embryos derived from ICSI or SCNT, after 4.5 days of culturing in vitro. Scale bar, 100 μm I. Preimplantation development rates in the tdTomato+ and tdTomato- embryos derived from ICSI or SCNT. The efficiency was calculated based on the number of 2-cell embryo that have been divided into tdTomato+ and tdTomato- groups. Error bars, s.d., n ≥ 3.
Wnt β-catenin signalling is unaffected in Slc7a5-null embryos, but is required for Slc7a5 transcription and β-catenin loss induces ISR gene Trib3 (A-B) (A,a1) Wnt β-catenin target Axin2 strongly expressed in littermate, (a2, a3) in TS, and (B, b1) in Scl7a5-null embryos (b2, b3) in TS (n=2 littermates, n=6/6 Slc7a5-null embryos).Figure 7(A-B) (A,a1) Wnt β-catenin target Axin2 strongly expressed in littermate, (a2, a3) in TS, and (B, b1) in Scl7a5-null embryos (b2, b3) in TS (n=2 littermates, n=6/6 Slc7a5-null embryos). Data information: White dashed lines indicate position of sections. Scale bars 200 µm (C, d1) Axin2 transcripts in embryo trunk explant exposed to DMSO (C, c1, n=12/12) were reduced in Wnt-c59 exposed explants (D, d1, n=14/14). Images in (c1, d1) show TS through explants (data from 3 independent embryo explant experiments). (E, F) Slc7a5 transcripts in embryo trunk explant exposed to DMSO (E, e1, n=9/10) or Wnt-c59 (F, f1, n=15/15). Images in (e1, f1) show TS through explants (data from 3 independent embryo explant experiments). Data information: Scale bars = 100 µm (G, H) Slc7a5 expression in control T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryo (G, g1) and in TS (g2, g3), and in T-Cre;Ctnnb1flLOF/Δ mutant embryo where Slc7a5 transcripts are absent in posterior neural tube (the region of T-cre recombination, indicated with white arrowhead in H) and shown in TS in (h2, h3) (n= 0/6 littermate and n=6/6 mutant embryos). Data information: White dashed lines indicate position of sections. Scale bars, 200 µm (I, J) Trib3 is lacking in T-Cre;Ctnnb1flLOF/Δ heterozygous control embryos at E7.75 (I), caudal region dissected from the same embryo (white dashed box in I) (i1) and viewed in TS (i2); Trib3 detected in homozygous T-Cre;Ctnnb1flLOF/Δ embryo (J), in caudal region dissected from the same embryo (white dashed box in J) (j1) and shown in TS in caudal epiblast and remnant primitive streak (ps) (j2) (n= 3/3 T-Cre;Ctnnb1flLOF/Δ embryos and 14 littermate controls). Trib3 was also detected in the more rostral forming neural tube at this early stage (seen in J) and so in cells not directly experiencing β-catenin loss; this may reflect the failure to form paraxial mesoderm, which provides signals that promote and support neurogenesis. Data information: White dashed lines indicate position of sections. Scale bars all sections 50 µm. (K-M) At E9.5 Trib3 was not detected in T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryos (K, k1) and in TS ( k2, k3), but was detected in homozygous T-Cre;Ctnnb1flLOF/Δ mutant embryos (L-m3) in patches of cells in neural tube (nt) (L - l2), gut (g) (l3) and somites (s) (m2, arrowheads) (n=9/9 T-Cre;Ctnnb1flLOF/Δ embryos and 12 littermate controls). Data information: White dashed lines indicate position of sections. Scale bars, all sections 50 µm.
Evaluation of the lower-limit processing capabilities.Figure 3A) Schematic representation of the experimental design with a 1:2 dilution series of a HeLa batch lysate starting from 10 μg down to 5 ng. The distribution of samples across the 96-well plate is shown. The dilution series was prepared in four replicates and samples were injected from lowest to highest concentration. For the four highest concentrated samples 1 μg material was injected, whereas for sub-microgram samples the entire sample was used. The average number of quantified proteins per sample as well as the corresponding sum iBAQ intensities are shown with error bars from the 4 replicates. B) Schematic representation of the experimental design of processing low numbers of HeLa cells. Series of decreasing cell numbers were prepared from 10.000 to 10 cells, in 8 replicates. The average number of quantified proteins per sample as well as the corresponding sum iBAQ intensities are shown with error bars from the eight replicates.
Figure 5Deletion of the N-terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres. U2OS stably expressing vector (V), WT Myc-CTC1 and Myc-∆700N were treated with or without HU and co-stained with Myc (red) and TRF2 (green) antibodies. Boxed areas are amplified in inserts to indicate CTC1/TRF2 colocalization (yellow).Scale bars: 10 μm. Quantification of percent of cells with >3 CTC1/TRF2 colocalization foci were from three independent experiments. In each experiment, >150 cells from each sample were analyzed.    ∆700N does not affect CST complex formation. HEK293T cells were co-transfected with Myc- CTC1 or Myc-∆700N, His6-STN1 and HA-TEN1. Co-IP was performed with Myc antibody to pulldown His6- STN1 and HA-TEN1. Full-length Myc-CTC1 was prone to degradation during immunoprecipitation.      ∆700N retains RAD51 interaction. HEK293T cells were co-transfected with Myc-CTC1 or Myc-∆700N, His6- STN1, HA-TEN1 and Flag-RAD51 or vector control (V), and treated with HU (2 mM, 16 h). Co-IP was performed with Flag antibody to pulldown Myc-CTC1.   DNA fiber analysis of CTC1 depleted U2OS cells with RNAi-resistant WT or ∆700N re-expression. Mean values in each sample are listed at the top of the graph. N, the number of cells analyzed in each condition, ***
Figure 4(A) Schematic diagram of the structures of Rv2626c (up). 293T cells were co-transfected with Flag-TRAF6 together with GST-vector or GST-Rv2626c and its mutant constructs, subjected to GST pulldown, followed by IB with αFlag. WCLs were used for IB with αGST, αFlag or αActin (down). (B) At 12 hr post-transfection with mammalian Myc-Rv2626c constructs together with Flag-TRAF6 and 293T cells treated with several Tat-CBS2 peptide for 12 h (10 µM) for 6h, subjected to IP with αMyc, followed by IB with αFlag. WCLs were used for IB with αMyc, αFlag or αActin. (C) Schematic diagram of the structures of TRAF6 (up). 293T cells were co-transfected with Myc-Rv2626c together with GST-vector or GST-TRAF6 and its mutant constructs, subjected to GST pulldown, followed by IB with αMyc. WCLs were used for IB with αGST, αMyc or αActin (down). (D) 293T cells were co-transfected with Flag-TRAF6, Myc-Rv2626c as indicated doses and HA-ubiquitin (left), HA-K48-linked ubiquitin (middle), HA-K63-linked ubiquitin (right) and then IP with αFlag, followed by IB with αHA. WCLs were used for IB with αFlag , αMyc, or αActin. (E) BMDMs were pretreated with 2.5 μg/ml rRv2626c for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αTRAF6, IB with ubiquitin, K48-linked ubiquitin, or K63-linked ubiquitin. WCLs were used for IB with αHis, αTRAF6, or αActin.
Differential gene expression analysis indicates ISG20 as candidate nuclease.Figure 2A3A expressing dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell and treated 7 days after infection with 300 U/ml IFNα (+ IFNα) or without (- IFNα). Microarray data was analyzed for upregulated nucleases after 6 or 72 hours of treatment, respectively. dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated 9 days later with 300 U/ml IFNα (B) or 200 U/ml IFNγ (C) for indicated times (h: hours). Expression of indicated genes was analyzed by qRT-PCR relative to TATA-box binding protein (TBP) mRNA (n=6 biological replicates of two independent experiments). dHepaRG cells were infected with HBV at an MOI of 100 virions/cell or not infected and treated 7 days later with 300 U/ml IFNα for 6 hours or 200 U/ml IFNγ for 24 hours. ISG20 mRNA levels were measured by qRT-PCR relative to TBP mRNA (n=6 biological replicates of two independent experiments). dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated with 300 U/ml IFNα from day 7 to 9 p.i. or 200 U/ml IFNγ from day 7 to 10 p.i. Primary human hepatocytes were infected with HBV at an MOI of 100 virions/cell and treated with 500 U/ml IFNα or 200 U/ml IFNγ from day 4 to 6 p.i. Expression of ISG20 was detected by western blot.