EMBO/soda-nel huggingface dataset:
Tousled-like kinase 2 regulates recovery from a DNA damage-induced G2 arrest (2016)
Figure 3

Figure 3. Depletion of Tlk2 leads to altered kinetics of the DNA damage response. A) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release. Cells were fixed at the indicated times after adriamycin-induced DNA damage and stained for DAPI and γH2AX. Scale bar indicates 10 μm. B) Quantification of cells in 3A. Each dot represents the total signal of γH2AX in the nucleus of a single cell. Bars indicate the mean and error bars indicate the SD of the data points in each condition. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (NS for P>0.05, **** for P≤0.0001). C) U2OS cells were synchronized in G2 and harvested at the indicated times after induction of adriamycin-induced DNA damage with or without Tlk2-depletion. Samples were analyzed by western blotting for the indicated proteins. D) U2OS cells were synchronized in G2 and damaged with 6 Gy of IR. Samples were taken at the indicated times after induction of damage with or without Tlk2-depletion. Samples were analyzed by western blot for the indicated proteins. Band signal intensity for Asf1A and Asf1B was measured and corrected for Ponceau S staining. E) U2OS cells were transfected with the indicated siRNA’s and treated as in 2A. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05). F) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for the indicated protein levels. Lanes were all present on the same blot but pasted together for comparison as indicated by the lines. G) Cells were synchronized in S- or G2-phase by a single thymidine block. Damage was induced by 0.5 μM Adriamycin for one hour and cells were allowed to incubate for 16 hours afterwards. Samples were fixed at the indicated cell cycle stages and stained for Asf1A-pS166. Nuclear fluorescence intensity was measured relative to DAPI. Whiskers represent 5-95% of data points. H) Cells were treated and analyzed as in 2E. Induction of FLAG-Asf1A-4D was induced using doxycycline. Error bars represent SD, n=5. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05). I) U2OS cells were synchronized in G2 by a single thymidine block and where indicated treated with 0.5 μM Adriamycin for one hour and after the indicated time points were pre-extracted and fixed. Cells were stained for total levels of Histone H3 and quantified relative to DAPI. Scale bar indicates 10 μm. Whiskers represent 5-95% of data points. J) FLAG-Asf1A-4D inducible cells were treated as in 3I and expression was induced using doxycycline where indicated. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (**** for P≤0.0001).
Tousled-like kinase 2 regulates recovery from a DNA damage-induced G2 arrest (2016)
Figure 4

Figure 4. Depletion of Tlk2 leads to a loss of recovery competence. A) U2OS cells were treated as in 2B after which mRNA was isolated and analyzed by qPCR. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05). B) Tlk2Δ 9.3.1 cells were treated and analyzed as in 3A. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01). C) U2OS cells were synchronized in G2 and harvested or synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins. D) U2OS cells were transfected with the indicated siRNA's, synchronized in G2 and damaged with 6 Gy of IR. Cells were harvested 24 hours later and the mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01). E) Cells were treated as in 4C. F) U2OS cells were transfected with the indicated siRNA's, arrested in G2 with Adriamycin and induced to recover by addition of caffeine. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01). G) U2OS cells were synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins. H) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt were thymidine synchronized, damaged in G2 and harvested 24 hours afterwards. Tetracycline was either absent (off), added from the start of the experiment (early) or for 16 hours after induction of DNA damage (late). I) Cells were treated as in 5G and recovery was induced by addition of caffeine for 8 hours. Mitotic index was analyzed by FACS and normalized to the control. Error bars represent SD of three independent experiments. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01). J) Model of Tlk2/Asf1A-dependent recovery competence. Red circle denotes phosphorylation event at S166, white circles denote histones. See text for further details.
Cooperation of LIM domain-binding 2 (LDB2) with EGR in the pathogenesis of schizophrenia (2021)
Figure 1

Figure 1: Expression of Ldb2 in the specific regions in the mouse brains

A. Schematic representation of the human LDB2 protein. Positions for the variants found in bipolar patients are also shown. The shorter isoform predicted from the splice variant (Tran et al, 2006) is shown below.

B. Multiple alignments of the N-terminal regions of the human and mouse LDB family. Please note that the N-terminal regions are highly diverged between LDB1/Ldb1 and LDB2/Ldb2. The bar indicated the position for the peptide for antibody production.

C. Tissue distribution of the Ldb2 protein. Representative western-blot image is shown. Yellow asterisks indicate uncharacterized bands.

D. Expression of Ldb2 in the cerebral cortex. DAPI-stained nuclei visualized (right) in the cerebral cortex.

E. Expression of Ldb2 in the amygdala. LA; lateral amygdala, BLA; basolateral amygdala, Ce; central nucleus of amygdala.

F-G. Expression of Ldb2 in neurons. The merged images obtained with Ldb2 (stained in green) and NeuN (stained in red) antibodies are shown for cerebral cortex layers I-VI and the amygdala (F). Please note that most of Ldb2-positive cells are NeuN-positive. G. NeuN-positive cells/Ldb2-positive cells (left) and Ldb2-positive cells/NeuN-positive cells were counted in cerebral cortex layers I-VI. Data are shown as means ± SD (10 independent view fields/group).
Cooperation of LIM domain-binding 2 (LDB2) with EGR in the pathogenesis of schizophrenia (2021)
Figure 2

Figure 2: Behavioral characterization of the Ldb2 KO mice

A-H. Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in various behavioral tests. Home cage-activity test (A). Voluntary locomotor activities in the home cages were monitored for a week. Data of dark (left) and light (right) phases are indicated. Open-field test (B), Acoustic startle response (C), Prepulse-inhibition test (D), Morris water-maze test (E) where data in hidden test (escape latency and moving speed, path length and no movement time) and probe test (path length) are shown, and Y-maze test (F). Fear-conditioning test (G). Results of contextual (left) and cued (right) tests are shown. Cumulative values are shown at the bottom. Hot-plate test (H). *; p < 0.05, **; p < 0.01, ***; p < 0.001 [Tukey test except for F and G (bottom) where t-test was used]. The error bars represent S.E.M.
Cooperation of LIM domain-binding 2 (LDB2) with EGR in the pathogenesis of schizophrenia (2021)
Figure 7

Figure 7: LDB2/Ldb2 forms complexes with known transcription regulators

A-E. Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates are shown. LDB2 vs. LDB2 (A), LDB2 vs. LHX2 (B), LDB2 vs. LMO2/3/4 (C), LDB2 vs. SSBP2/3/4 (D), and LDB2 vs. RLIM (E). LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.

F. Coimmunoprecipitation of endogenous Ssbp3 and Lmo3 with Ldb2. Immunocomplexes from mouse brain lysate using an antibody against Ldb2, Lmo3 or Ssbp3, or a control antibody were analyzed by western blotting using an anti-Ldb2 (left) or anti-Ssbp3 (right) antibody.
Cooperation of LIM domain-binding 2 (LDB2) with EGR in the pathogenesis of schizophrenia (2021)
Figure 8

Figure 8: Possible cooperation of LDB2 and EGR transcription factors revealed by ChIP-seq

A. Human neurosphere (bottom) induced from iPS cells (top) from a healthy control subject.

B. Sequence motifs identified by the MEME-ChIP program. Shown are the top three motifs identified in the LDB2 ChIP-seq peak region (summit ± 100 bp of the top 2000 ChIP-seq peaks) (See Fig EV4 for more motifs).

C. Overlap of ChIP-seq peaks of LDB2 in neurosphere cells and ChIP-seq peaks of EGR1 in human cultured cell lines ECC1 and K562, at ARC (See Appendix Table S3 for the dataset used for the analysis).

D. Genome-wide pattern of overlap between the ChIP-seq peaks of LDB2 in neurosphere cells and the ChIP-seq peaks of EGR1 in the ECC1 cell line. Fold enrichment (log2) of ChIP-seq reads against the input reads were calculated for each ChIP-seq peak region (summit ± 1,000 bp) in each category (neurosphere specific, common, and ECC1 specific) and displayed in a heatmap by ngs.plot (Shen et al, 2014). See Fig EV4 for the overlap between the ChIP-seq peaks of LDB2 and EGR1 in other cell lines.

E. Annotation of ChIP-seq peaks. ChIP-seq peaks of LDB2 in the neurosphere cells (all, neurosphere specific, and common, shown in D were analyzed by the PAVIS program (Huang et al, 2013).
Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1 (2022)
Figure 1

Figure 1. Loss of UBP12 and UBP13 caused growth defects and decreased BR sensitivity in Arabidopsis

Growth phenotypes of the wild-type (WT, Col-0), bri1-null mutant, and two independent ubp12i/ubp13 double mutant lines grown on 1/2MS medium supplemented with 10 μM DEX (DEX medium) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution (0.1% [v/v] EtOH) for additionally 8 days. Scale bars: 10 mm.

Phenotype of the WT and ubp12i/ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days. Scale bars: 10 mm.

Hypocotyl length relative to the mock control of seedlings (B). Experiments were done in triplicate (n >15 seedlings for each line).

Western blot (WB) analysis of BES1 dephosphorylation after BL treatment with the α-BES1 antibody. Total proteins were isolated from 6-day-old seedlings grown on DEX medium in the light, either in the presence of 100 nM BL or mock. PEPC was used as a loading control.

Percentage of dephosphorylated BES1 relative to the total BES1 from (D) (n = 6 biological replicates).

Data information: data are presented as means ± SD (C and E). **P<0.01 (one-way ANOVA and post-hoc Tukey's test) compared with WT under each condition (C). P<0.01 (one-way ANOVA and post-hoc Tukey's test) (E).
Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1 (2022)
Figure 2

Figure 2. UBP12 and UBP13 negatively regulated BRI1 protein degradation

Western blot analysis of BRI1 protein amount in ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from 18-day-old plants grown on 1/2MS medium supplemented with 10 μM DEX (+) or not (-).

Western blot analysis of BRI1 protein level in concanamycin A (ConcA)-treated plants. Eighteen-day-old plants were grown on DEX medium and exposed to 1 μM ConcA for 4 h before total protein isolation.

Data information: PEPC was used as a loading control. The values shown above each lane indicate the abundance of the BRI1 protein relative to that of WT without DEX treatment (A) or to that of WT without ConcA treatment (B).
Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1 (2022)
Figure 3

Figure 3. UBP13 interacts with and deubiquitinates BRI1

Split-ubiquitin yeast two-hybrid assay revealing the UBP13 and BRI1 interactions. The indicated constructs were cotransformed into yeast cells. BRI1-Cub with Nub, Nub-CIPK8, and Nub-UBP13 with an empty vector were used as negative controls. Transformants were streaked onto solidified medium supplemented with X-gal. Blue patch indicates positive interaction. Cub, the C-terminal half of ubiquitin; Nub, the N-terminal half of ubiquitin.

UBP13 interaction with BRI1 in plants. BRI1-Myc and FLAG-UBP13 were coexpressed in N. benthamiana leaves and coimmunoprecipitation was done on solubilized microsomal proteins with α-Myc antibody beads. The association of BRI1-UBP13 was detected by western blot with an α-FLAG antibody after α-Myc IP (Top).

Deubiquitinating activity of UBP13 against K48- and K63-linked ubiquitination. Diubiquitins linked through K48 or K63 (K48 Di-Ub or K63 Di-Ub) were incubated alone or with GST-UBP13 or GST-UBP13C207S for 18 h. Deubiquitination of the diubiquitins was analyzed by western blot with the α-ubiquitin (α-Ub, P4D1) antibody. The presence of GST-UBP13/UBP13C207S recombinant proteins was confirmed by the α-GST antibody.

UBP13 deubiquitination of K63-ubiquitinated BRI1 in vitro. Polyubiquitinated MBP-BRI1CD was generated by incubation with GST-PUB13 (Ub reaction) and then with GST-UBP13 or GST-UBP13C207S (DUB reaction-MBP pull-down, input). After in vitro deubiquitination, MBP-BRI1CD was purified by an MBP pull-down to analyze the BRI1CD ubiquitination (DUB reaction-MBP pull-down, SE). The ubiquitinated proteins were detected by western blot with an α-ubiquitin antibody (FK2). Ubiquitin chain specificity was detected with an α-K63Ub antibody (Apu3). The presence of recombinant proteins was confirmed by the α-GST and α-MBP antibodies.

Western blot analysis of BRI1 ubiquitination on plants expressing pBRI1:BRI1-mCitrine complementing the bri1 mutant in either the UBP12/UBP13 (BRI1-mCit/bri1) or ubp12i/ubp13 (BRI1-mCit/bri1/ubp12i/ubp13) background. BRI1-mCitrine proteins were isolated from 18-day-old plants grown on DEX medium and then immunoprecipitated with α-GFP antibody beads from solubilized microsomal proteins. Ubiquitinated BRI1 and basal BRI1 proteins were detected by the α-ubiquitin (P4D1) and α-GFP antibodies, respectively.

Quantification of BRI1 ubiquitination profiles from (E) (n = 3 biological replicates). The ubiquitinated BRI1 fraction was normalized to the total immunoprecipitated BRI1 detected by the α-GFP antibody.

Data information: data in (F) are presented as means ± SD.
Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1 (2022)
Figure 4

Figure 4. The ubiquitination-deficient BRI1 restores the growth defects of ubp12i/ubp13

Growth phenotype comparison of BRI1-mCit/bri1/ubp12i/ubp13 and BRI125KR-mCit/bri1/ubp12i/ubp13. The indicated transgenic lines were grown on DEX medium for 18 days. Scale bars: 10 mm.

Western blot analysis monitoring the BRI1 protein accumulation in BRI125KR-mCit/bri1/ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from the roots of the lines indicated (A). PEPC was used as a loading control.

Hypocotyl length relative to the mock control. Seedlings of the indicated lines were grown on DEX medium containing 100 nM BL or mock in the light for 6 days (Appendix Fig S7

A). Experiments were done in triplicate (n > 15 seedlings for each line).

Data information: data in (C) are presented as means ± SD. P<0.05 (one-way ANOVA and post-hoc Tukey's test).
Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1 (2022)
Figure 5

Figure 5. The vacuolar targeting of BRI1 is accelerated in ubp12i/ubp13

A-D Analysis of the BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 (A) or BRI125KR (C) tagged with mCitrine (mCit) in either bri1/UBP12/UBP13 or bri1/ubp12i/ubp13 background. Six-day-old seedlings were grown on DEX medium, and kept in the dark for 2 h in the presence of 50 μM CHX before imaging. Confocal images of epidermal cells from root meristem are shown. Scale bars: 10 μm. Measurements of the relative PM/intracellular BRI1-mCit fluorescence intensity. For each line, at least 55 and 25 cells from over 25 and 15 seedlings were measured in (B) and (D), respectively.

Pulse-chase AFCS uptake experiments in WT and ubp12i/13 plants. Six-day-old seedlings were grown on DEX medium and pulsed with 40 µM AFCS for 40 min followed by a 30-min chase before imaging. Scale bars: 10 μm.

Quantification of AFCS fluorescence intensity in (E) (n > 30 ROIs from 7 to 9 seedlings for each line).

Data information: measurements shown in box plots are the first and third quartiles and are split by the medians, whiskers extending 1.5-fold interquartile range beyond the box (B, D, and F). Data points are plotted as dots. n.s., not significant, *P<0.05, **P<0.01 (one-way ANOVA and post-hoc Tukey's test).
Vesicle sub-pool organization at inner hair cell ribbon synapses (2018)
Figure 1

Fig. 1: The number of RA-SVs increases at inhibited OtofPga/Pga ribbon synapses

(A) Schematic summary of tomogram analysis (not drawn to scale). SVs-pools: ribbon-associated (RA) SVs: first row, ≤ 80 nm from the ribbon surface (R, with filaments: dark green; without filaments: light green), membrane-proximal (MP) SVs: ≤ 50 nm membrane-to-membrane distance in the first row from AZ-membrane (blue) and ≤ 100 nm from the PD (pink) (with tethers: orange; non-tethered: yellow; docked: light orange). Analysis parameter also described in [24,25,45].

(B-G) Tomographic 3D reconstructions of the RA-SV pool at wild-type (B-D) and OtofPga/Pga (E-G) in inhibitory (inhib, B,E), resting (rest, C,F) and stimulatory (stim, D,G) conditions (color codes see A). MP-SVs, PD and AZ-membrane in transparent. Scale bar, 100 nm.

(H,I) Comparable mean SV number per ribbon in wild-type at the distal (Ds., H) and the proximal (Pr., I) part of the ribbon. More SVs are observed at inhibitory OtofPga/Pga ribbons compared to inhibitory wild-type synapses at both ribbon parts, and further at OtofPga/Pga synapses under rest in the Pr. part. Data are presented as mean ± SEM; the red dotted line highlights the wild-type mean SV number per ribbon under rest as a reference point; one-way ANOVA followed by Tukey's test; ** p < 0.01, * p < 0.05. Values: Appendix Table S3A, S4A.

(J,K) Representative virtual sections of wild-type (J) and OtofPga/Pga (K) ribbon synapses under inhibition. Counted RA-SVs are marked with white asterisks. Magnification, 12,000x; scale bar, 100 nm.

(L, M) Exemplary virtual sections of wild-type and OtofPga/Pga under inhib (L,M), rest (L',M') and stim (L'',M''). At OtofPga/Pga SVs are densely packed. Blue arrowheads indicate proteinaceous filaments between wild-type SVs. Magnification 12,000x; scale bar, 100 nm.
Vesicle sub-pool organization at inner hair cell ribbon synapses (2018)
Figure 3

Fig 3: Fractions of RA-SV sub-pools at the distal and proximal ribbon part at wild-type and OtofPga/Pga ribbon synapses

(A,B) Depicted are the fractions of SVs without filaments, ribbon-attached SVs (Ri.), interconnected SVs (In.) and SVs with Ri. + In. per ribbon, at (A) the distal (Ds.) and (B) the proximal (Pr.) part of the ribbon. Data are represented as mean ± SEM of wild-type and OtofPga/Pga IHCs under inhib, rest and stim. (A) Upon stimulation, in the Ds. part at wild-type, the fraction of SVs without filaments reduces, but of SVs with Ri. + In. increases significantly. At OtofPga/Pga IHCs the fraction of SVs without filaments is reduced when comparing stim vs. inhib. The fraction of interconnected SVs increases with stimulation compared to inhibition and rest. In the mutant (inhib), the fraction of interconnected SVs decreases compared to wild-type (inhib), and Ri. + In. SVs reduce vs. wild-type (stim). (B) The fraction of SVs without filaments increased at inhibited OtofPga/Pga synapses. The red dotted lines in (A) and (B) represent the mean fraction of RA-SVs/ribbon part of resting wild-type for each defined sub-pool. P-values are calculated by one-way ANOVA followed by Tukey's test; p-value of the trend is shown, * p < 0.05, *** p < 0.001, **** p < 0.0001 and ns p > 0.05. Values in Appendix Table S3 and S4.

(C) Schematic summary: the distal (Ds.) ribbon (R) part, at wild-type (left panel) and OtofPga/Pga (right panel) ribbon synapses upon stimulation. Not drawn to scale; SVs with filaments (dark green, blue filaments) and without filaments (light green).
Vesicle sub-pool organization at inner hair cell ribbon synapses (2018)
Figure 4

Fig 4: Shorter filaments in the RA-SV pool upon stimulation at wild-type and OtofPga/Pga ribbon synapses

(A) Representative wild-type virtual section under rest depicts SVs with ribbon-attached filaments (Ra.fil., red arrowheads) and one interconnector (Intercon., purple arrowhead), around the ribbon (R). Magnification 12,000x; scale bar, 40 nm.

(A'-A''') Example 3D reconstruction of the RA-SVs with filaments (green) in resting wild-type. Larger average length is found for ribbon-attached filaments (Ra.fil., red) vs. SV interconnectors (Intercon., purple). For clear visualization, the ribbon (R) is transparent and the same SV pair is shown in a front (A'), side (A'') and top view (A'''). Scale bar, 50 nm.

(B) Increased mean length of ribbon-attached filaments vs. SV interconnectors in all conditions for both genotypes. P-values are calculated by K-W test corrected for multiple-comparison; **** p < 0.0001.

(C) The cumulative probability distribution of RA-SVs plotted against the individual filament length. The graph illustrates shorter filaments in wild-type and OtofPga/Pga ribbon synapses upon stimulation.

(D) Decreased mean filament length upon stimulation in wild-type and OtofPga/Pga ribbon synapses. Resting OtofPga/Pga exhibit the shortest mean filament length. P-values are calculated by K-W test corrected for multiple-comparison; * p < 0.05, ** p < 0.01, **** p < 0.0001 and ns p > 0.05.

The red dotted lines in (B) and (D) represent the mean RA-filament length in resting wild-type for each sub-pool.