Journal: bioRxiv

Article Title: PIEZO1 and PIEZO2 Blade domains are differentially required for channel localization and function

doi: 10.1101/2024.04.06.588398

Figure Lengend Snippet: A) Immunocytochemistry of PIEZO2 ( 1–644 ) encompassing the Distal Blade (anti-GFP antibody) along with an Endoplasmic Reticulum marker ( ER-mCherry ) (anti-RFP antibody). B) Immunocytochemistry of AQP3-tGFP (1st row) and AQP3-THU3-tGFP (anti-turboGFP antibody) along with cis Golgi Marker (2nd row, Giantin-mCherry) or an ER (ER-mCherry) (3rd row, anti-RFP antibody); the inset highlights the ring-shaped accumulations of the chimeric protein.

Article Snippet: Samples were then incubated ON at 4°C with a dilution of 1/2,000 anti-GFP chicken antibody (Aves Lab; GFP-1020), or a dilution of 1/1,000 anti-turboGFP chicken antibody (Origene; TA150075) respectively, and a dilution of 1/1,000 anti-RFP rabbit antibody (Rockland; 600-401-379).

Techniques: Immunocytochemistry, Marker

Journal: Scientific Reports

Article Title: Neuronal morphology and synaptic input patterns of neurons in the intermediate nucleus of the lateral lemniscus of gerbils

doi: 10.1038/s41598-023-41180-8

Figure Lengend Snippet: Primary antibodies used for histology.

Article Snippet: MAP2 , Chicken , Polyclonal , AMCA , 1:1000 , OriGene , TA336617.

Techniques:

Journal: Physiological Reports

Article Title: Combinatory effects of si RNA ‐induced myostatin inhibition and exercise on skeletal muscle homeostasis and body composition

doi: 10.1002/phy2.262

Figure Lengend Snippet: Effect of myostatin inhibition (after myostatin knockout [ Mstn −/− ], AAV ‐mediated overexpression of myostatin propeptide [ AAV Prop] and si RNA ‐mediated Mstn knockdown) on myostatin propeptide mRNA levels and serum protein levels. (A) Quantitative RT‐PCR analysis of Mstn mRNA levels in Mstn −/− extensor digitorum longus ( EDL ) and soleus muscles (primers targeting exons 2/3). n = 5 per group, * P < 0.05 (B) Quantitative RT‐PCR analysis of myostatin propeptide m RNA levels in Mstn −/− muscle (primers targeting exons 1/2). n = 5 per group (C) Quantitative RT‐PCR analysis of M stn m RNA levels in AAV Propeptide treated tibialis anterior ( TA ) muscle (primers targeting exons 2/3). n = 6 per group * P < 0.05 (D) Quantitative RT‐PCR analysis of myostatin propeptide RNA levels in AAV Propeptide‐treated TA muscle (primers targeting exon 1/2). n = 6 per group, * P < 0.05 (E) Immuno‐ PCR analysis to determine serum concentration of myostatin propeptide ( MYOPRO ) from Mstn −/− mice. n = 5 per group, * P < 0.05. (F) Immuno‐ PCR analysis to determine serum concentration of MYOPRO following si RNA ‐mediated Mstn knockdown ± exercise. n = 7 per group, * P < 0.05 significantly different from control group. C = control group, T = training group, si = treatment with si RNA , siT = training + si RNA . KO = Mstn −/− mice, WT = wild‐type mice. Values are presented as means ± SD .

Article Snippet: The following capture antibodies and recombinant proteins were used: goat polyclonal antihuman follistatin antibody (AF669, R&D Systems GmbH, Wiesbaden‐Nordenstadt, Germany), chicken polyclonal antihuman myostatin propeptide antibody (RD183057050, BioVendor), recombinant human follistatin (669FO/CF, R&D Systems), recombinant human myostatin propeptide (RD172058100, BioVendor GmbH, Heidelberg, Germany).

Techniques: Inhibition, Knock-Out, Over Expression, Quantitative RT-PCR, Concentration Assay

Journal: Physiological Reports

Article Title: Combinatory effects of si RNA ‐induced myostatin inhibition and exercise on skeletal muscle homeostasis and body composition

doi: 10.1002/phy2.262

Figure Lengend Snippet: Primer sequences

Article Snippet: The following capture antibodies and recombinant proteins were used: goat polyclonal antihuman follistatin antibody (AF669, R&D Systems GmbH, Wiesbaden‐Nordenstadt, Germany), chicken polyclonal antihuman myostatin propeptide antibody (RD183057050, BioVendor), recombinant human follistatin (669FO/CF, R&D Systems), recombinant human myostatin propeptide (RD172058100, BioVendor GmbH, Heidelberg, Germany).

Techniques:

Journal: Physiological Reports

Article Title: Combinatory effects of si RNA ‐induced myostatin inhibition and exercise on skeletal muscle homeostasis and body composition

doi: 10.1002/phy2.262

Figure Lengend Snippet: Serum measurements of follistatin, myostatin propeptide, and leptin

Article Snippet: The following capture antibodies and recombinant proteins were used: goat polyclonal antihuman follistatin antibody (AF669, R&D Systems GmbH, Wiesbaden‐Nordenstadt, Germany), chicken polyclonal antihuman myostatin propeptide antibody (RD183057050, BioVendor), recombinant human follistatin (669FO/CF, R&D Systems), recombinant human myostatin propeptide (RD172058100, BioVendor GmbH, Heidelberg, Germany).

Techniques:

Journal: Inhalation toxicology

Article Title: Neuroglobin mitigates mitochondrial impairments induced by acute inhalation of combustion smoke in the mouse brain

doi: 10.3109/08958378.2014.902147

Figure Lengend Snippet: (A) Representative immunofluorescent images of mitochondrial spreads (wild type) reacted with cytochrome c (green) and synapsin 1 (red) antibodies (40X magnification) are shown. (B) Graphical representation of ImageJ quantification of cytochrome c (green) and synapsin (red) positive particles. (C) Western blotting analysis of crude mitochondrial preparations probed for the mitochondrial outer membrane, voltage dependent anion channel protein 1 (VDAC1) and the inter-membrane space cytochrome c protein. Detection of the synaptosomal protein, synapsin 1, confirmed the presence of synaptosomal component in cortical mitochondria preparations. Neuroglobin was detected in preparations from Ngb-tg but not the wild type cortex. (D) Representative contrast phase image of uniformly distributed crude mitochondrial preparations in XF24 plates (6 μg/well) loaded for oxygen consumption analyses (20X magnification).

Article Snippet: Antibodies: heme oxygenase-1 monoclonal (sc-136960, Santa Cruz), VDAC1 (sc-8828, Santa Cruz), neuroglobin (RD181043050, BioVendor).

Techniques: Western Blot

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: S100A11 interacts with RAD51 at sites of DSB repair. (A) HaCaT cells synchronized in S phase were treated with bleomycin (12.5 µg/ml) for 30 min and released in fresh medium for 90 min or were untreated (control) followed by immunostaining with antibodies against S100A11 (green) and γH2AX (red). Nuclear DNA was detected using DAPI (blue). Bar, 10 µm. (B) S phase HaCaT cells that were analyzed by laser scanning microscopy for S100A11 (green) and RAD51 (red) at sites of DNA damage 2 hour after bleomycin treatment. Bar, 10 µm. An area marked by a rectangle (light blue) in the overlay image (merge) is shown enlarged below the respective image. The intensities of the immunofluorescences in one cell derived from the RAD51 signal (red) and the S100A11 signal (green) are shown in a linescan (left side of the enlarged overlay). (C) Quantification of the colocalization of S100A11 with γH2AX (white Whisker box), RAD51 (gray Whisker box) and γH2AX/RAD51 (black Whisker box) in HaCaT cells after DSB induction. Colocalization events were determined in nuclei of cells treated as described in A. Thirty nuclei from 2 independent experiments were analyzed. Data are displayed as mean values (±SD). (D) Co-immunoprecipitation (CoIP) experiments between S100A11 and RAD51. A specific anti-S100A11 antibody precipitated RAD51 from whole cell extracts of S phase HaCaT cells treated with bleomycin (12.5 µg/ml) (lane 4) or untreated S phase cells (lane 6). In a control, an unspecific antibody used in CoIP experiments was unable to precipitate RAD51 from the same extracts (lanes 3 and 5). (E) The authenticity of the RAD51/S100A11 interaction was confirmed by pull-down with overexpressed proteins. U2OS cells were transfected with a plasmid encoding FLAG-S100A11 alone or together with a His-RAD51 encoding plasmid followed by synchronization in S phase and induction of DSBs by bleomycin treatment. Pull-down (pd) was carried out from cell extracts of the transfected cells for His-tagged proteins binding to Talon resins, and immunoblotting was performed using the antibodies as indicated. As loading control (load), 5% of the cell extract was used.

Article Snippet: Anti-S100A11 chicken polyclonal antibody (BP4007; Acris Antibodies) at 1:50, anti-RAD51 rabbit polyclonal antibody (ABE257; Millipore) at 1:250, anti-6xHis mouse monoclonal antibody (ab18184; Abcam) at 1:100, anti-53BP1 rabbit polyclonal antibody (sc-22760; Santa Cruz) at 1:50, anti-γH2AX (Ser139) (clone JBW301; 05-636; Millipore) at 1:750, and anti-γH2AX rabbit serum (kindly provided by P. Hemmerich, FLI Jena) at 1:100 were used in 2- or 3-color immunofluorescence staining as primary antibodies which were detected with species-specific secondary antibodies linked to fluorescein, Cy3, or Cy5 (Dianova) all at 1:200.

Techniques: Immunostaining, Laser-Scanning Microscopy, Derivative Assay, Whisker Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Binding Assay, Western Blot

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: S100A11 is functionally required for complete DSB repair. (A) Down-regulation of S100A11 in HaCaT cells treated with specific siRNA against S100A11 (lane 2: S100A11 siRNA#6, lane 3: S100A11 siRNA#7) was confirmed by immunoblotting using a specific antibody against S100A11. As control, HaCaT cells transfected with nonspecific control (nsc) siRNA (lane1) were used. GAPDH served as loading control. (B) Experimental setup. HaCaT cells were transfected with the indicated siRNA and subsequently synchronized in S phase by double-thymidine block. DNA damage was induced by treatment with 12.5 µg/ml bleomycin for 30 min. Cells were harvested after different time points (2 and 8 h repair) for immunostaining against S100A11 and γH2AX. (C) γH2AX foci persist in damaged S100A11 knock-down cells. Control represents cells without bleomycin treatment. The number of γH2AX foci per cell is shown as Whisker graphs. Mean values represent 3 independent experiments with n > 30 cells analyzed for each condition. (D) Analysis of the percentage of individual cells possessing γH2AX foci. HaCaT cells were treated as described in B. Mean values represent analysis of n > 90 cells for each condition in 3 independent experiments. (E) RAD51 foci (white) persist upon DSB damage after S100A11 knock-down. HaCaT cells were treated as described in B and analyzed by immunostaining against RAD51 and S100A11. Nuclear DNA was detected using DAPI (blue). Bar, 10 µm. (F) Quantification of percentage of individual cell showing RAD51 foci. Mean values represent analysis of n > 85 cells for each condition in 3 independent experiments. * P < 0.05, *** P < 0.001.

Article Snippet: Anti-S100A11 chicken polyclonal antibody (BP4007; Acris Antibodies) at 1:50, anti-RAD51 rabbit polyclonal antibody (ABE257; Millipore) at 1:250, anti-6xHis mouse monoclonal antibody (ab18184; Abcam) at 1:100, anti-53BP1 rabbit polyclonal antibody (sc-22760; Santa Cruz) at 1:50, anti-γH2AX (Ser139) (clone JBW301; 05-636; Millipore) at 1:750, and anti-γH2AX rabbit serum (kindly provided by P. Hemmerich, FLI Jena) at 1:100 were used in 2- or 3-color immunofluorescence staining as primary antibodies which were detected with species-specific secondary antibodies linked to fluorescein, Cy3, or Cy5 (Dianova) all at 1:200.

Techniques: Western Blot, Transfection, Blocking Assay, Immunostaining, Whisker Assay

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: S100A11 stimulates the strand exchange activity of RAD51. (A) left panel Scheme of the strand exchange reaction between circular ssDNA and linearized dsDNA. right panel Strand exchange by human RAD51 requires Ca2+. RAD51, derived from 2 distinct purification procedures, (lanes 2–3 and 5–6: 3 µM) was incubated with 24 µM circular ΦX174 ssDNA in strand exchange buffer containing 2 mM of either magnesium or calcium acetate for 15 min at 37°C followed by incubation with 2.4 µM RPA for 5 min and addition of 24 µM linearized ΦX174 dsDNA to initiate strand exchange reaction for 2 h at 37°C. Lane M: constructed joint molecule DNA product derived from ssDNA/dsDNA annealing used as marker (B) left panel S100A11 enhances RAD51-mediated strand exchange. RAD51 (lanes 4–6: 3 µM) alone or with S100A11 (lane 5: 2 µM, lane 6: 4 µM) was incubated as described in (A) in strand exchange buffer containing calcium acetate (2 mM). As negative control, S100A11 (lane 7: 4 µM) was incubated alone. The joint molecule product (jm) was visualized by GelStar staining. right panel Quantification of S100A11-stimulated joint molecule formation by RAD51. Average values of 3 independent experiments are shown with standard derivation. (C) Dialysis of S100A11 abrogated the stimulating effect of S100A11 on RAD51 activity. RAD51 (lanes 4–8 and 10) together with undialyzed S100A11 (lane 5), S100A11 dialyzed in EGTA containing buffer (lanes 7–9), or S100A11 dialyzed in buffer without EGTA (lane 10), was incubated with 24 µM circular ΦX174 ssDNA in strand exchange buffer containing 2 mM of either magnesium or calcium acetate for 15 min at 37°C followed by incubation with 2.4 µM RPA for 5 min and addition of 24 µM linearized ΦX174 dsDNA to initiate strand exchange reaction for 2 h at 37°C. *P < 0.05.

Article Snippet: Anti-S100A11 chicken polyclonal antibody (BP4007; Acris Antibodies) at 1:50, anti-RAD51 rabbit polyclonal antibody (ABE257; Millipore) at 1:250, anti-6xHis mouse monoclonal antibody (ab18184; Abcam) at 1:100, anti-53BP1 rabbit polyclonal antibody (sc-22760; Santa Cruz) at 1:50, anti-γH2AX (Ser139) (clone JBW301; 05-636; Millipore) at 1:750, and anti-γH2AX rabbit serum (kindly provided by P. Hemmerich, FLI Jena) at 1:100 were used in 2- or 3-color immunofluorescence staining as primary antibodies which were detected with species-specific secondary antibodies linked to fluorescein, Cy3, or Cy5 (Dianova) all at 1:200.

Techniques: Activity Assay, Derivative Assay, Purification, Incubation, Construct, Marker, Negative Control, Staining

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: A S100A11 mutant without Ca2+-binding impairs DSB repair. (A) left panel Immunostaining of U2OS cells for γH2AX in cells expressing recombinant S100A11. Cells expressing S100A11ΔCa display significantly increased γH2AX levels 8 h after DSB induction. Cells transfected with a plasmid encoding S100A11wt or S100A11ΔCa, respectively, were synchronized in S phase, followed by treatment with 12.5 µg/ml bleomycin for 30 min. Cells were harvested 8 h after induction of DNA damage and analyzed by immunostaining against γH2AX (white). Nuclear DNA was detected using DAPI (blue). Bar, 10 µm. right panel Quantification of the percentage of cells showing γH2AX foci after S100A11wt (n = 88) or S100A11ΔCa (n = 99) transfection. The results of 3 independent experiments are presented. Data are shown as the mean ±SD. (B) Recombinant S100A11ΔCa expression increases RAD51 foci persistence. left panel U2OS cells were treated as above and analyzed by immunostaining against RAD51 (white). Nuclear DNA was detected using DAPI (blue). Bar, 10 µM. right panel Quantification of percentage of individual cells showing RAD51 foci. Mean values represent analysis of cells expressing S100A11wt (n = 94) or S100A11ΔCa (n = 99) of 3 independent experiments. (C) Expression of S100A11wt (lane 2) or S100A11ΔCa (lane 3) in pcDNA4-transfected U2OS cells was confirmed by immunoblotting against S100A11. An empty plasmid was used for mock-transfection of U2OS cells (lane 1). GAPDH served as loading control. (D) S100A11ΔCa mutant failed to interact with RAD51. U2OS cells were transfected with a plasmid encoding FLAG-S100A11 (wild-type or ΔCa mutant, respectively) alone or together with a His-RAD51 encoding plasmid followed by synchronization in S phase and induction of DSBs by bleomycin treatment. Pull-down (pd) was carried out from cell extracts of the transfected cells using Talon resins to precipitate His-tagged proteins together with interacting partners. For analysis of the pulled-down interacting proteins, immunoblotting was performed using the antibodies as indicated. As loading control (load), 5% of the cell extract was used. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Anti-S100A11 chicken polyclonal antibody (BP4007; Acris Antibodies) at 1:50, anti-RAD51 rabbit polyclonal antibody (ABE257; Millipore) at 1:250, anti-6xHis mouse monoclonal antibody (ab18184; Abcam) at 1:100, anti-53BP1 rabbit polyclonal antibody (sc-22760; Santa Cruz) at 1:50, anti-γH2AX (Ser139) (clone JBW301; 05-636; Millipore) at 1:750, and anti-γH2AX rabbit serum (kindly provided by P. Hemmerich, FLI Jena) at 1:100 were used in 2- or 3-color immunofluorescence staining as primary antibodies which were detected with species-specific secondary antibodies linked to fluorescein, Cy3, or Cy5 (Dianova) all at 1:200.

Techniques: Mutagenesis, Binding Assay, Immunostaining, Expressing, Recombinant, Transfection, Plasmid Preparation, Western Blot

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: Downregulation of S100A11 results in restricted recombination capacity, chromosomal aberrations, and reduced cell viability. (A) S100A11 knock-down leads to a significant decrease in SCE. (top) Representative examples show multiple SCEs (left nsc siRNA, right S100A11 siRNA; white arrows) and (bottom) quantification of SCE per metaphase of 2 independent experiments. HaCaT cells with or without S100A11 siRNA were treated with bleomycin (12.5 µg/ml) for 30 min, allowed to recover for 2 h and transferred to medium containing 25 µM BrdU. After 4 h, 0.2 µg/ml Colcemid was added in fresh medium for 18 h to collect cells in metaphase. At least 40 metaphases per data point were analyzed. Error bars represent standard errors of the mean. (B-D) S100A11 knock-down leads to a significant increase of chromosomal abberations. HaCaT cells transfected with S100A11 siRNA or nsc siRNA were treated with 12.5 µg/ml bleomycin for 30 min. After further culture for 20 h, 0.1 µg/ml Colcemid was added for another 6 h to enrich cells in metaphase. The number of chromosome breaks (B) and complex chromosome aberrations (CCA) (C) are shown. Chromosome breaks and CCA were scored in n = 160 metaphase cells for each condition for 2 independent experiments. Error bars represent the standard errors of means. (D) Representative metaphase nuclei containing chromosomal aberrations as detected in S100A11-depleted HaCaT cells. Arrows (red) point to chromatid breaks; arrowheads (blue) point to radial figures. These aberrations are typical for DSBs occurred after DNA replication. (E) S100A11 knock-down leads to a significant loss of cell viability after bleomycin treatment. HaCaT cells transfected with specific S100A11 siRNA or control nsc siRNA for 72 h were treated with bleomycin (12.5 µg/ml) for 30 min. After this, medium was exchanged and the cells cultured for another 10 days. Then, the number of colonies formed was determined using Clono-Counter software in 3 independent experiments. HaCaT cells transfected with the control nsc siRNA without bleomycin treatment were used as control. * P < 0.05, ** P < 0.01, ***P < 0.001.

Article Snippet: Anti-S100A11 chicken polyclonal antibody (BP4007; Acris Antibodies) at 1:50, anti-RAD51 rabbit polyclonal antibody (ABE257; Millipore) at 1:250, anti-6xHis mouse monoclonal antibody (ab18184; Abcam) at 1:100, anti-53BP1 rabbit polyclonal antibody (sc-22760; Santa Cruz) at 1:50, anti-γH2AX (Ser139) (clone JBW301; 05-636; Millipore) at 1:750, and anti-γH2AX rabbit serum (kindly provided by P. Hemmerich, FLI Jena) at 1:100 were used in 2- or 3-color immunofluorescence staining as primary antibodies which were detected with species-specific secondary antibodies linked to fluorescein, Cy3, or Cy5 (Dianova) all at 1:200.

Techniques: Transfection, Cell Culture, Software