Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Time‐dependent changes in systolic blood pressure ( SBP ) following angiotensin II (Ang II ) infusion in mice. A , Time course of SBP levels in Ang II –stimulated BubR1 low‐expression ( BubR1 L/L ) mice and wild‐type ( BubR1 +/+ ) mice from days 0 to 6 of Ang II infusion. n=10 BubR1 +/+ mice; n=11 BubR1 L/L mice. B , SBP levels at day 1 and day 6 of Ang II infusion. * P <0.01 vs BubR1 +/+ mice.

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Histological analysis of tissues from BubR1 low‐expression ( BubR1 L/L ) mice and wild‐type ( BubR1 +/+ ) mice after 1 week of angiotensin II (Ang II ) infusion. A , Representative Sirius red‐stained kidney sections. Magnification, ×40; n=10 BubR1 +/+ mice; n=8 BubR1 L/L mice; scale bar, 100 μm. B , Perivascular fibrotic lesion area in the kidney, quantified using morphometry. * P <0.01 vs BubR1 +/+ mice. C , Representative Elastica van Gieson–stained sections of the thoracic aorta. Magnification, ×40; n=12 BubR1 +/+ mice; n=11 BubR1 L/L mice; scale bar, 100 μm. D , Area of aorta media, quantified using morphometry. * P <0.01 vs BubR1 +/+ mice. E , Representative Masson trichrome–stained sections of the heart. Magnification, ×40; n=12 BubR1 +/+ mice; n=11 BubR1 L/L mice; scale bar, 100 μm. F , Ejection fraction, evaluated by echocardiographs; n=12 control mice; n=11 BubR1 L/L mice. * P <0.01 vs BubR1 +/+ mice.

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Expressing, Staining, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Immunohistochemistry of kidneys from BubR1 +/+ and BubR1 L/L mice before and after 1 week of angiotensin II (Ang II ) infusion. A through C , Images of representative kidney sections that were immunostained for BubR1 ( A ), Ang II receptor type 1 ( AGTR 1) ( B ), or Ang II receptor type 2 ( AGTR 2) ( C ). Magnification, ×40; scale bar, 50 μm. BubR1, AGTR 1, and AGTR 2 expression levels in kidney with or without BubR1 reduction, with or without angiotensin II (Ang II ) stimulation (n=5/group). * P <0.01. kpxls indicates kilo pixels; n.s., not significant.

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Immunohistochemistry, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: In vitro assay of BubR1 reduction in human renal proximal tubule cells ( RPTC s). A , BubR1 levels in RPTC s that were treated with siBubR1 or scrambled control si RNA (3 biological replicates each). *P <0.01. B , Angiotensin II (Ang II ) receptor type 1 ( AGTR 1) expression levels in RPTC s with or without BubR1 reduction, with or without 10 μmol/L Ang II stimulation for 24 hours (n=3/group). * P <0.01. siBubR1 indicates short interfering RNA targeting BubR1 .

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: In Vitro, Control, Expressing, Small Interfering RNA

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Expression levels of Nox 4, JNK , and phospho‐ JNK in human renal proximal tubule cells ( RPTC s), with or without angiotensin II (Ang II ) stimulation. A , Representative Western blot of Nox 4, JNK , and phospho‐ JNK / JNK in siBubR1‐ or scramble si RNA control–treated RPTC s, with or without Ang II stimulation. B through D , Quantified expression levels of Nox 4 ( B ), JNK ( C ), and phospho‐ JNK / JNK ( D ) from the blot in ( A ) (n=6/group). * P <0.01. JNK indicates Jun N‐terminal kinase; Nox4, nicotinamide adenine dinucleotide phosphate oxidase‐4; n.s., not significant; p‐JNK, phospho‐JNK; siBubR1, short interfering RNA targeting BubR1 .

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Expressing, Western Blot, Control, Small Interfering RNA

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Immunohistochemistry of kidneys from BubR1 +/+ and BubR1 L/L mice before and after 1 week of angiotensin II (Ang II ) infusion. Representative immunohistochemistry sections of Nox4 ( A ) and JNK ( B ) expression. Scale bar, 100 μm. Nox4 and SAPK / JNK expression levels in kidney with or without BubR1 reduction, with or without Ang II stimulation (n=5/group). Quantified expression levels of Nox 4 ( C ), and SAPK / JNK ( D ). * P <0.01. JNK indicates Jun N‐terminal kinase; Nox4, nicotinamide adenine dinucleotide phosphate oxidase‐4; n.s., not significant; SAPK, stress‐activated protein kinase.

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Immunohistochemistry, Expressing

Journal: Nature communications

Article Title: The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

doi: 10.1038/ncomms6563

Figure Lengend Snippet: Figure 1 | Recruitment of Cdc20 to kinetochores requires BubR1. (a) Schematic of protocol used to synchronize cells and deplete specific proteins using RNAi. (b) Cells depleted of the indicated proteins were stained for CRESTand Cdc20 and BubR1 or Mad2 to check for depletion efficiency. Scale bar, 5 mm. (c) Quantification of Cdc20, BubR1 and Mad2 kinetochore levels in cells treated with the indicated RNAi oligos. The fluorescence from the three z-stacks (200 nm apart) encompassing the bulk kinetochore fluorescent intensity was used and normalized to the CREST signal. Cells were co-stained for Mad2 or BubR1 to ensure that only cells with efficient depletion were analysed. At least 80 kinetochore pairs from eight cells were quantified and the mean and s.e.m. is indicated. (d) Stable HeLa cell line expressing Venus-Cdc20 was treated with the indicated RNAi oligos and the localization of Venus-Cdc20 followed by time-lapse microscopy. Scale bar, 10 mm. (e) Quantification of Venus-Cdc20 at kinetochores in the movies in (d). The signal was quantified from a single z-section and in the frame right after NEBD. A total number of 50 kinetochores from 10 cells were analyzed for each condition and the mean and s.e.m. indicated. An unpaired t-test was performed for statistical analysis (****Po0.0001) and compared with the control-treated cells.

Article Snippet: The following antibodies were used for western blot, immunofluorescence or immunoprecipitation as indicated: a-tubulin 11H10 (rabbit, Cell Signaling) 1:100 IF, b-actin AC-15 (mouse, Abcam) 1:5,000 WB, APC1 A301– 653A (rabbit, Bethyl) 1:500 WB, APC3 35/CDC27 (mouse, BD Biosciences) 1:250 WB, APC4 (mouse, raised against a C-terminal peptide) 5 mg IP, APC7 A302–551A (rabbit, Bethyl) 1:1,000 WB, Bub3 clone 31 (mouse, BD Transduction Laboratories) 1:500 WB, BubR1 A300–995A (rabbit, Bethyl) 1:500 WB, BubR1 A300–386A (rabbit, Bethyl) 1:200 IF, Cdc20 AR12 (mouse, Millipore) 1:200 IF/5 mg IP, Cdc20 E-7 (mouse, Santa Cruz Biotechnology) 1:500 WB, Cdc20 A301–180A (rabbit, Bethyl) 1:1,000 WB, CREST (human, Antibodies Inc.) 1:400 IF, FLAG M2 (mouse, Sigma) 1:5,000 WB, GFP clones 7.1 and 13.1 (mouse, Roche) 1:5,000 WB /1:200 IF, Mad2 from G. Kops (rabbit) 1:200 IF, Mad2 A300–301A (rabbit, Bethyl) 1:1,000 WB, p31 rabbit antibody was generated using full-length p31 as antigen and affinity purified.

Techniques: Staining, Expressing, Time-lapse Microscopy, Control

Journal: Nature communications

Article Title: The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

doi: 10.1038/ncomms6563

Figure Lengend Snippet: Figure 3 | The internal Cdc20 binding site of BubR1 recruits Cdc20 to kinetochores. (a) Schematic of human BubR1 and the location of Cdc20 binding sites and Bub3 binding site as well as the pseudo-kinase domain. Alignment of the region encompassing residues 530–535 of human BubR1 is shown on top and the different constructs used are indicated below. The BubR1 KEN/AAA has the first KEN-box mutated to AAA. (b) Stable HeLa cell lines expressing the different Venus-BubR1 siRNA resistant constructs were used to determine the domains in BubR1 required for Cdc20 kinetochore localization. In brief, cells were treated with a control RNAi oligo (Luciferase) or a BubR1 RNAi oligo and then arrested in mitosis using nocodazole treatment and the proteasome inhibitor, MG132. BubR1 RNAi-treated cells were complemented with the indicated Venus-BubR1 constructs. Cells were stained for BubR1, CREST and Cdc20. Scale bar, 5 mm (c,d). The kinetochore levels of BubR1 (c) and Cdc20 (d) normalized to CREST in control and BubR1-depleted cells. At least 80 kinetochore pairs from eight different cells were analysed, and the mean and s.e.m. are indicated. (e) The level of Cdc20 at kinetochores in cells complemented with the indicated Venus-BubR1 constructs was determined. Only cells with endogenous levels of BubR1 at kinetochores were used for this analysis. At least 80 kinetochore pairs from eight different cells were analysed, and the mean and s.e.m. are indicated. (f) Binding of Cdc20 and Cdc20 4A to BubR1 516–715 was determined by binding 10 mg recombinant FLAG-HA-BubR1 516–715 to FLAG affinity beads and incubating these with increasing concentrations of Strep-His tagged Cdc20 or Cdc20 4A expressed and purified from HEK293 cells. The beads were washed and bound proteins were eluted and analysed by western blot. (g) Quantification of western blot in (f) using Licor technology. Experiment in (f,g) is representative of two independent experiments.

Article Snippet: The following antibodies were used for western blot, immunofluorescence or immunoprecipitation as indicated: a-tubulin 11H10 (rabbit, Cell Signaling) 1:100 IF, b-actin AC-15 (mouse, Abcam) 1:5,000 WB, APC1 A301– 653A (rabbit, Bethyl) 1:500 WB, APC3 35/CDC27 (mouse, BD Biosciences) 1:250 WB, APC4 (mouse, raised against a C-terminal peptide) 5 mg IP, APC7 A302–551A (rabbit, Bethyl) 1:1,000 WB, Bub3 clone 31 (mouse, BD Transduction Laboratories) 1:500 WB, BubR1 A300–995A (rabbit, Bethyl) 1:500 WB, BubR1 A300–386A (rabbit, Bethyl) 1:200 IF, Cdc20 AR12 (mouse, Millipore) 1:200 IF/5 mg IP, Cdc20 E-7 (mouse, Santa Cruz Biotechnology) 1:500 WB, Cdc20 A301–180A (rabbit, Bethyl) 1:1,000 WB, CREST (human, Antibodies Inc.) 1:400 IF, FLAG M2 (mouse, Sigma) 1:5,000 WB, GFP clones 7.1 and 13.1 (mouse, Roche) 1:5,000 WB /1:200 IF, Mad2 from G. Kops (rabbit) 1:200 IF, Mad2 A300–301A (rabbit, Bethyl) 1:1,000 WB, p31 rabbit antibody was generated using full-length p31 as antigen and affinity purified.

Techniques: Binding Assay, Construct, Expressing, Control, Luciferase, Staining, Recombinant, Western Blot

Journal: Nature communications

Article Title: The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

doi: 10.1038/ncomms6563

Figure Lengend Snippet: Figure 5 | The IC20BD is required for SAC silencing. (a) Stable HeLa cells were depleted of endogenous BubR1 or treated with control RNAi oligo (Luciferase). BubR1 knockdown was complemented by expression of Venus-BubR1, Venus-BubR1 DBub3 or Venus-BubR1 DBub3/D490–560 as indicated and mitotic progression was followed by time-lapse microscopy. Each circle represents a single cell analysed (at least 40 cells were analysed for each condition) and the median (m) is shown as a red bar. Statistical analysis was performed using a Mann–Whitney test. (b) Stable HeLa cell lines were depleted of endogenous BubR1 and complemented by expression of Venus-BubR1 or Venus-BubR1 D490–560 as indicated. Cells were arrested in mitosis with 100 nM nocodazole before filming and subsequently treated with 0.5 mM reversine to silence the SAC. Each circle represents a single cell analysed and at least 120 cells were analysed per condition. Red bars indicate means (m) and a t-test was used for statistical analysis. (c) Representative still images from (b). Scale bar, 10 mm. (d,e) Venus-Cdc20 was purified from a stable HeLa cell line arrested in mitosis with nocodazole. The beads were incubated with increasing concentrations of recombinant FLAG-HA-BubR1 516–715 at room temperature for 1 h and afterwards washed. The binding of endogenous BubR1 and Mad2 to Venus-Cdc20 was analysed by western blot analysis and quantified using Licor technology. Representative of two independent experiments. (f) HeLa cells were transfected with the indicated BubR1 constructs and before filming 200 nM taxol was added. The time from NEBD to mitotic exit was measured by analysing the time-lapse movies and at least 50 cells were analysed per condition. Medians (m) are shown as red bars and a Mann–Whitney test was used for statistical analysis.

Article Snippet: The following antibodies were used for western blot, immunofluorescence or immunoprecipitation as indicated: a-tubulin 11H10 (rabbit, Cell Signaling) 1:100 IF, b-actin AC-15 (mouse, Abcam) 1:5,000 WB, APC1 A301– 653A (rabbit, Bethyl) 1:500 WB, APC3 35/CDC27 (mouse, BD Biosciences) 1:250 WB, APC4 (mouse, raised against a C-terminal peptide) 5 mg IP, APC7 A302–551A (rabbit, Bethyl) 1:1,000 WB, Bub3 clone 31 (mouse, BD Transduction Laboratories) 1:500 WB, BubR1 A300–995A (rabbit, Bethyl) 1:500 WB, BubR1 A300–386A (rabbit, Bethyl) 1:200 IF, Cdc20 AR12 (mouse, Millipore) 1:200 IF/5 mg IP, Cdc20 E-7 (mouse, Santa Cruz Biotechnology) 1:500 WB, Cdc20 A301–180A (rabbit, Bethyl) 1:1,000 WB, CREST (human, Antibodies Inc.) 1:400 IF, FLAG M2 (mouse, Sigma) 1:5,000 WB, GFP clones 7.1 and 13.1 (mouse, Roche) 1:5,000 WB /1:200 IF, Mad2 from G. Kops (rabbit) 1:200 IF, Mad2 A300–301A (rabbit, Bethyl) 1:1,000 WB, p31 rabbit antibody was generated using full-length p31 as antigen and affinity purified.

Techniques: Control, Luciferase, Knockdown, Expressing, Time-lapse Microscopy, MANN-WHITNEY, Incubation, Recombinant, Binding Assay, Western Blot, Transfection, Construct

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells (40.87 ± 9.95 arbitrary units [a.u.] [WT] vs. 39.48 ± 6.04 a.u. [LUZP1 KO]). n = 3. P = 0.54 (Mann–Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio (1.00 [1 st lane, control] vs. 1.13 ± 0.24 [2 nd lane] vs. 1.01 ± 0.44 [3 rd lane] vs. 1.08 ± 0.73 [4 th lane]). n = 4. P = 0.49 (Kruskal–Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting. Representative confocal micrographs of co‐cultures of Venus‐LUZP1‐expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm. Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells (control, 21.14 ± 16.80 a.u. [WT] vs. 3.10 ± 1.72 a.u. [LUZP1 KO]; calyculin A, 25.24 ± 10.54 a.u. [WT] vs. 20.65 ± 5.62 a.u. [LUZP1 KO]; washout, 22.09 ± 7.90 a.u. [WT] vs. 7.92 ± 4.01 a.u. [LUZP1 KO]). ** P < 0.01 (Mann–Whitney U test). Bars and error bars represent the mean ± SD. n = 3. Representative immunoblot of WT, LUZP1 KO, and Venus‐LUZP1‐expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A (WT, 1.00 [control] vs. 1.40 ± 0.06 [calyculin A] vs. 1.14 ± 0.33 [washout]; KO, 0.09 ± 0.04 [control] vs. 1.49 ± 0.06 [calyculin A] vs. 0.81 ± 0.99 [washout]; REV, 2.06 ± 1.78 [control] vs. 1.82 ± 1.50 [calyculin A] vs. 1.80 ± 1.14 [washout]). n = 3. Bars and error bars represent the mean ± SD. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Knock-Out, MANN-WHITNEY, Standard Deviation, In Vitro, Phosphorylation Assay, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c β/δ, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20‐kDa regulatory subunit (M20). PP1c β/δ represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c β/δ and MLC. Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells (28.68 ± 9.60 arbitrary units [a.u.] [WT] vs. 25.04 ± 9.47 a.u. [LUZP1 KO]). P = 0.09 [Mann–Whitney U test]. n = 3. Bars and error bars represent the mean ± standard deviation (SD). Co‐immunoprecipitation of HA‐PP1c β/δ and GFP‐LUZP1. LUZP1 binds to PP1c β/δ. IB, immunoblotting. In vitro MLC phosphorylation assay using 1 μg GST‐PP1c β/δ in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the di‐phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 upregulated ppMLC/MLC levels in a dose‐dependent manner (1.00 [1 st lane, control] vs. 1.27 ± 0.33 [2 nd lane] vs. 1.76 ± 0.68 [3 rd lane] vs. 2.53 ± 1.65 [4 th lane] vs. 2.93 ± 2.45 [5 th lane]). n = 3 or 6. ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 μg GST‐PP1c β/δ, 100 ng GST‐Merlin, 2 pg p21‐activated kinase 1 (PAK1), and 5 μg GST‐LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 upregulated pMerlin/Merlin levels (0.23 ± 0.15 [1 st lane] vs. 1.00 [2 nd lane, control] vs. 0.32 ± 0.17 [3 rd lane] vs. 0.97 ± 0.42 [4 th lane] vs. 1.25 ± 0.39 [5 th lane]). n = 4 or 9. * P < 0.05, ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 3 rd lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Binding Assay, Knock-Out, MANN-WHITNEY, Standard Deviation, Immunoprecipitation, Western Blot, In Vitro, Phosphorylation Assay

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Box plots with dot density plots showing the ratio of the apical area/basal area in co‐cultures of Venus‐LUZP1‐expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells (REV, 0.65 ± 0.16 [control] vs. 0.90 ± 0.18 [nocodazole] vs. 0.64 ± 0.16 [washout]; KO, 1.30 ± 0.17 [control] vs. 1.07 ± 0.13 [nocodazole] vs. 1.32 ± 0.19 [washout]). ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). The solid lines represent the medians, and the boxes represent the interquartile ranges. The error bars extending from the box represent the data within 1.5 times of the interquartile range. Representative confocal micrographs of co‐cultures of LUZP1‐expressing wild‐type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di‐phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm. Bar plots with dot density plots showing that ppMLC levels within CRs were significantly downregulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment (WT, 21.43 ± 6.96 arbitrary units [a.u.] [control] vs. 17.67 ± 5.40 a.u. [nocodazole] vs. 20.84 ± 7.19 a.u. [washout]; KO, 8.74 ± 1.71 a.u. [control] vs. 8.67 ± 1.89 a.u. [nocodazole] vs. 7.96 ± 2.35 a.u. [washout]). n = 3. ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). Bars and error bars represent the mean ± standard deviation (SD). In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, 1 μg GST‐protein phosphatase 1c β/δ (PP1c β/δ), and 0–5 μg GST‐LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1‐mediated inhibition of PP1c β/δ (1.00 [1 st ‐lane, control] vs. 1.42 ± 0.59 [2 nd ‐lane] vs. 1.72 ± 0.76 [3 rd ‐lane] vs. 1.99 ± 0.56 [4 th ‐lane] vs. 1.14 ± 0.37 [5 th ‐lane] vs. 2.87 ± 1.51 [6 th ‐lane] vs. 2.74 ± 1.19 [7 th ‐lane] vs. 2.50 ± 0.88 [8 th ‐lane]). n = 6. * P < 0.05 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Expressing, Knock-Out, Standard Deviation, In Vitro, Phosphorylation Assay, Inhibition

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification

Journal: Advanced Science

Article Title: Scutellarin Rescued Mitochondrial Damage through Ameliorating Mitochondrial Glucose Oxidation via the Pdk‐Pdc Axis

doi: 10.1002/advs.202303584

Figure Lengend Snippet: SG promoted mitochondrial aerobic respiration and regulated mitochondrion‐dependent apoptosis via inhibiting PDK2 as the target. A) Docking analysis of SG spatially bond around ATP‐binding domain of PDK2. B) Docking analysis of SG spatially bond around lipoamide‐binding domain of PDK2. C) Docking analysis of SG spatially bond around CoA‐binding domain of PDK2. D) The percent inhibition ratio of SG against PDK2 compared with DCA. E) Western blot of SK‐N‐SH cell line with stable knockdown of PDK2 (shPDK2). F) Mitochondrial membrane potential evaluation of vehicle SK‐N‐SH cell and shPDK2 SK‐N‐SH cell during mitochondrial OXPHOS damage. G) The western blot of the DARTS experiment. H) The western blot of the CETSA experiment. I) The results of the co‐IP‐MS experiment. J) The results of the Lip‐MS experiment. K) Cell mitochondrial stress test of vehicle SK‐N‐SH cell and shPDK2 SK‐N‐SH cell. L) Cell mitochondrial stress test of shPDK2 SK‐N‐SH cell during mitochondrial OXPHOS damage and SG administration. M) OCRs for basal respiration, ATP‐linked respiration, maximal respiration, and spare respiratory capacity of shPDK2 SK‐N‐SH cell during mitochondrial OXPHOS damage and SG administration. N) the western blot of mitochondria‐dependent apoptosis‐related proteins. O) The western blot of pyroptosis‐related proteins. P) Flow cytometry to compare apoptosis ratio of vehicle SK‐N‐SH cells and shPDK2 SK‐N‐SH cells under mitochondrial damage and SG administration. Data are presented as mean ±SEM by paired two‐tailed Student's t ‐test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, model versus control group, # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001, SG versus model group.

Article Snippet: The percentage inhibition ratio of SG, against PDK2 (Carna Cat. No. 10–140) and PDK4 (Carna Cat. No. 10–125), were tested with dichloroacetic acid (DCA) as the positive control.

Techniques: Binding Assay, Inhibition, Western Blot, Membrane, Co-Immunoprecipitation Assay, Flow Cytometry, Two Tailed Test

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 and TG003 chemical structures. ( b ) Pharmacokinetic profile of TG693 after a single 30 mg kg −1 dose administered by subcutaneous injection in imprinting control region (ICR) mice. Data indicate the mean ± SEM (n = 3). ( c ) Recombinant CLK1 was incubated with the substrate peptide in the presence of the indicated concentrations of small molecules. Data represent the means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown. ( d ) TG693 competitive ATP inhibition is shown in Michaelis-Menten (left) and Hanes-Woolf (right) plots. CLK1 kinase activity was measured at the indicated concentrations of TG693 and ATP. Velocity was plotted versus [ATP] and [ATP]/velocity was plotted versus [ATP]. ( e ) Map of the inhibitory activities of TG693 on a kinase dendrogram. Percent inhibition by 1 μM TG693 was measured for a panel of 313 kinases. Red circles indicate the inhibited kinases and are sized according to percent inhibition. The illustration was reproduced courtesy of Cell Signaling Technology, Inc. ( www.cellsignal.com ).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Injection, Recombinant, Incubation, Inhibition, Activity Assay

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) SR protein phosphorylation was assessed in HeLa cells treated with TG693 and TG003 for 1 h. Lamin B served as a loading control. Uncropped images have been provided in . ( b , c ) Effect of TG693 on exon 31 skipping with the reporter plasmid. Transfected HeLa cells were incubated in the presence of TG693, TG003, or DMSO vehicle for 24 h. Reporter and endogenous CLK1 splicing was then analyzed by RT-PCR. GAPDH served as a control. The Splicing ratios were quantified by intensity analysis and normalized to GAPDH expression. Uncropped images have been provided in and , respectively. Data represent the means ± SD (n = 3).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Plasmid Preparation, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 bioavailability in the tibialis anterior (TA) muscle of ICR mice after oral administration of a single 30 mg kg −1 dose. Data represent the mean ± SEM (n = 3). ( b ) SR protein phosphorylation status in the TA muscle of ICR mice after oral administration. Lamin B served as a loading control. SRSF4 phosphorylation was quantified by densitometry. Uncropped images are provided in . Data represent means ± SD (n = 5). * p < 0.05. ( c,d ) Clk1 expression in the TA muscle, heart and diaphragm were analyzed by RT-PCR with a GAPDH internal control. Uncropped images are provided in and in , respectively. Data represent the means ± SD (n = 3). * p < 0.05.

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction