Journal: International Journal of Molecular Sciences

Article Title: Effects of Substance P and Neurokinin A on the Contractile Activity of Inflamed Porcine Uterus

doi: 10.3390/ijms232113184

Figure Lengend Snippet: The protein expression of NK1R ( A ) and NK2R ( B ) in the porcine myometrium from the control (CON), saline (SAL) and E. coli groups, estimated by Western blot analysis. Blots with representative bands for each group are shown in . Protein levels of studied receptors are expressed as the mean ± SEM of ratios of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ** p < 0.01, *** p < 0.001 compared between groups for the same type of receptor.

Article Snippet: Afterwards, membranes were incubated with rabbit polyclonal antibodies against NK1R (Novus Biologicals, Littleton, CO, USA, cat. no. NB 300-119, working dilution 1:500) and NK2R (LifeSpan BioSciences, Inc., Seattle, 161 WA, USA, cat. no. LS-C177106, working dilution 1:500) at 4 °C, for 18 h. In the next step, the membranes were washed in TBS-T buffer and then incubated (at 21 °C, for 1 h) with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC-HRP Kit, Vector Labs, Burlingame, CA, USA, cat. no. PK-6101, working dilution 1:3000).

Techniques: Expressing, Control, Saline, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Effects of Substance P and Neurokinin A on the Contractile Activity of Inflamed Porcine Uterus

doi: 10.3390/ijms232113184

Figure Lengend Snippet: Representative pictures of NK1R ( A – C ) and NK2R ( E – G ) in the myometrium of gilts from the control (CON), saline (SAL) and E. coli ( E. coli ) groups. Positive immunoreaction to NK1R is visible in myometrial muscular cells (MCM) and arteries ( A ) of the control ( A ), saline-( B ) and pathogens ( C )-injected uteri. Expression of NK2R display MCM and A in the control ( E ), saline-injected ( F ) and inflamed ( G ) uteri. Negative control (NC) for NK1R ( D ) and NK2R ( H ) was obtained following the use of rabbit normal IgG instead of primary antibodies.

Article Snippet: Afterwards, membranes were incubated with rabbit polyclonal antibodies against NK1R (Novus Biologicals, Littleton, CO, USA, cat. no. NB 300-119, working dilution 1:500) and NK2R (LifeSpan BioSciences, Inc., Seattle, 161 WA, USA, cat. no. LS-C177106, working dilution 1:500) at 4 °C, for 18 h. In the next step, the membranes were washed in TBS-T buffer and then incubated (at 21 °C, for 1 h) with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC-HRP Kit, Vector Labs, Burlingame, CA, USA, cat. no. PK-6101, working dilution 1:3000).

Techniques: Control, Saline, Injection, Expressing, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Effects of Substance P and Neurokinin A on the Contractile Activity of Inflamed Porcine Uterus

doi: 10.3390/ijms232113184

Figure Lengend Snippet: The influence of substance P (SP) on the contractile amplitude ( A , C ) and frequency ( B , D ) in the myometrium ( A , B ) and endometrium/myometrium ( C , D ) strips of gilts from the CON (grey bars), SAL (hatched bars) and E. coli (black bars) groups after using NK1R antagonist. Results were calculated for five gilts in each group. The effects of antagonist and individual SP doses are presented as percentages (mean ± SEM) of the baseline (pretreatment period) contractile amplitude and frequency, taken as 100% (horizontal lines). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the basal value in each group; AAA p < 0.001 compared between the CON and E. coli groups for the same treatment; B p < 0.05, BB p < 0.01, BBB p < 0.001 compared between the SAL and E. coli groups for the same treatment.

Article Snippet: Afterwards, membranes were incubated with rabbit polyclonal antibodies against NK1R (Novus Biologicals, Littleton, CO, USA, cat. no. NB 300-119, working dilution 1:500) and NK2R (LifeSpan BioSciences, Inc., Seattle, 161 WA, USA, cat. no. LS-C177106, working dilution 1:500) at 4 °C, for 18 h. In the next step, the membranes were washed in TBS-T buffer and then incubated (at 21 °C, for 1 h) with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC-HRP Kit, Vector Labs, Burlingame, CA, USA, cat. no. PK-6101, working dilution 1:3000).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Effects of Substance P and Neurokinin A on the Contractile Activity of Inflamed Porcine Uterus

doi: 10.3390/ijms232113184

Figure Lengend Snippet: The graphic shows the procedure for measuring contractility of uterine strips. Ach-acetylcholine; SP-substance P; NKA-neurokinin A; NK1R-neurokinin receptor subtype 1; NK2R-neurokinin receptor subtype 2; NK1R anta.-neurokinin receptor subtype 1 antagonist; NK2R anta.-neurokinin receptor subtype 2 antagonist. Concentrations of the used substances are given in moles.

Article Snippet: Afterwards, membranes were incubated with rabbit polyclonal antibodies against NK1R (Novus Biologicals, Littleton, CO, USA, cat. no. NB 300-119, working dilution 1:500) and NK2R (LifeSpan BioSciences, Inc., Seattle, 161 WA, USA, cat. no. LS-C177106, working dilution 1:500) at 4 °C, for 18 h. In the next step, the membranes were washed in TBS-T buffer and then incubated (at 21 °C, for 1 h) with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC-HRP Kit, Vector Labs, Burlingame, CA, USA, cat. no. PK-6101, working dilution 1:3000).

Techniques:

Journal: bioRxiv

Article Title: H2AFX and MDC1 Protect Genomic Integrity in Male Germ Cells by Promoting Recombination and Activation of the Recombination-Dependent Checkpoint

doi: 10.1101/235085

Figure Lengend Snippet: (A) Representative nuclear spread in the pachytene stage of 12.5 dpp H2afx -/- spermatocyte labeled with anti-SYPC3 and anti-SYCP1. Scale bar represents 5 μm. (B) Representative RT-PCR analysis of the expression of Spo11 splicing variant in the indicated genotypes of juvenile mice. Level of β-actin was used as loading control. (C) Representative image of synapsed X-Y chromosomes in wild-type spermatocytes at early-pachytene stage (panel I). SYCP3 (red) marks the axial element of the synaptonemal complex. X-Y chromosomes are encircled by the dotted square. Magnification in panel II shows the X-Y chromosomes. The arrow points to the PAR region. Magnification in panel III shows the FISH signal (green) identifying the PAR region. The white bar indicates the extension of the PAR-FISH signal that is measured as aproxy for chromatin loop length measure. The magnification bar in I represents 5μM. (D) Quantification of the average length of Y-PAR FISH signal among spermatocytes of indicated genotypes, with unsynapsed and synapsed Y-chromosome ( H2afx +/+ n= 3 mice; H2afx -/- n= 3 mice). Error bars = mean±SD; p= p value. (E) Representative early pachytene spermatocytes of indicated genotypes, labeled with anti-SYCP3 (I,V, IX), Y-PAR probe (II,VI, X) and anti-DMC1 antibody (III,VII,XI). White arrows indicate Y-chromosome, while asterisks indicate a non-specific signal of the FISH. Merge images (IV, VIII, XII) show the localization of DMC1 foci within the Y-PAR region. Three adult mice were tested for each genotype. (F) Quantification of nuclei with unsynapsed X-Y chromosomes at the pachynema stage in the indicated genotypes. Three adult mice were tested for each genotype. Error bars = mean±SD; p= p value.

Article Snippet: Sources and dilutions of antibodies for immunofluorescence were as follows: mouse anti-SYCP3 (Santacruz, sc-74569) 1:300; rabbit anti-SYCP3 (Novus Biologicals nb300-231) 1:300; rabbit anti-SYCP1 (Abcam) 1:200; rabbit anti-DMC1 (Santacruz, sc 22768) 1:100; rabbit anti-γH2AX (Cell signaling) 1:400-500; guinea pig anti-H1T (a gift from M.A.

Techniques: Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing, Variant Assay

Journal: bioRxiv

Article Title: H2AFX and MDC1 Protect Genomic Integrity in Male Germ Cells by Promoting Recombination and Activation of the Recombination-Dependent Checkpoint

doi: 10.1101/235085

Figure Lengend Snippet: (A-B) Representative images of nuclear spreads of the indicated stages, from wild-type and H2afx -/- spermatocytes labeled with anti-SYCP3 and anti-SYCP1 antibodies. Arrows point axes of unsynapsed chromosomes. Scale bar is 5 μ m. (C) Quantification of zygonema-like cells within zygonema class in 60 dpp mice of indicated genotypes ( H2afx +/+ n= 3, H2afx -/- n = 4 mice tested). (D) Quantification of pachynema-like cells within pachynema cells class in 60 dpp mice of indicated genotypes. ( H2afx +/+ n = 3; H2afx -/- n = 4 mice tested). Error bars are mean±SD; p= p value. (E) Westernblot analyses of testisinsoluble nuclear extracts from 60dpp mice testis of the indicated genotypes. Slow (phosphorylated) migrating forms of HORMAD1 are indicated by black and dark-grey arrows. One mouse per genotype was analyzed. Molecular mass is given in kilodaltons. (F) Representative images of nuclear spreads of spermatocytes from the indicated stages and genotypes, labeled with anti-SYCP3 and anti-pHORMAD1. Arrows point axes of unsynapsed homologs. (G) Representative image of H2afx -/- spread spermatocytes labeled with anti-SYCP3 (red), anti-pHORMAD1 (white) and FISH (green) to identify the Y-PAR probe. Right panel, magnifications of dotted squares regions representing the pattern of pHORMAD1 in synapsed (upper panel) and unsynapsed sex chromosomes (lower panels). White bars indicate the extension of Y and X chromosomes. Scale bars are 5 μ m.

Article Snippet: Sources and dilutions of antibodies for immunofluorescence were as follows: mouse anti-SYCP3 (Santacruz, sc-74569) 1:300; rabbit anti-SYCP3 (Novus Biologicals nb300-231) 1:300; rabbit anti-SYCP1 (Abcam) 1:200; rabbit anti-DMC1 (Santacruz, sc 22768) 1:100; rabbit anti-γH2AX (Cell signaling) 1:400-500; guinea pig anti-H1T (a gift from M.A.

Techniques: Labeling

Journal: bioRxiv

Article Title: H2AFX and MDC1 Protect Genomic Integrity in Male Germ Cells by Promoting Recombination and Activation of the Recombination-Dependent Checkpoint

doi: 10.1101/235085

Figure Lengend Snippet: (A) Bouins-fixed testis sections stained with Periodic acid-Schiff staining. Arrows indicate apoptotic cells in tubule at epithelial stage IV. Post-IV indicates a tubule at a stage past stage IV, where spermatocytes have completely disappeared. Pre-IV indicates a stage before stage IV. (B) Quantification of apoptotic cells in chromosome spread preparations of the indicated genotypes following TUNEL staining of chromosome spreads co-stained with anti-SYCP3 and anti-SYCP1 antibodies (three mice analyzed for each genotype). Error bars = mean±SD; p= p value. (C) Representative images of nuclear spreads of H2afx +/+ and H2afx -/- spermatocytes labeled with anti-SYCP3, anti-SYCP1 and TUNEL at zygonema and pachynema stages. Scale bar is 5 μ m.

Article Snippet: Sources and dilutions of antibodies for immunofluorescence were as follows: mouse anti-SYCP3 (Santacruz, sc-74569) 1:300; rabbit anti-SYCP3 (Novus Biologicals nb300-231) 1:300; rabbit anti-SYCP1 (Abcam) 1:200; rabbit anti-DMC1 (Santacruz, sc 22768) 1:100; rabbit anti-γH2AX (Cell signaling) 1:400-500; guinea pig anti-H1T (a gift from M.A.

Techniques: Staining, TUNEL Assay, Labeling

Journal: bioRxiv

Article Title: H2AFX and MDC1 Protect Genomic Integrity in Male Germ Cells by Promoting Recombination and Activation of the Recombination-Dependent Checkpoint

doi: 10.1101/235085

Figure Lengend Snippet: (A) Quantification of BRCA1 foci number in mice of the indicated genotypes. Each dot in the graph indicates the number of DMC1 foci per nucleus; the error-bars= means ± sd (see S1table for more details). Three mice analyzed for each genotype. (B) Representative images of localization of BRCA1 (red) along SYCP3-positive (green) chromosome axes of spermatocytes at zygonema, in the indicated genotypes. (C) Quantification of DMC1 foci number in the indicated genotypes. Three mice analyzed for each genotype (see S1 table for more details). (D) Quantification of MSH4 foci number in mice of the indicated genotypes. Each dot in the graph 5C and 5D indicates the number of foci per nucleus; error-bars= means ± sd; p= p value. (see S1 table for more details). (E) Representative images of nuclear spreads of indicated genotypes labeled with anti-SYCP3 and anti-MSH4 antibodies. Scale bar represents 5 μ m. (F) Representative images of nuclear spreads of indicated genotypes labeled with anti-SYCP3 and anti-MSH4 antibodies. Enlarged images are magnifications of dotted areas. The white arrows point to MSH4 foci at representative synaptic forks. Scale bar is 5 μ m. (G) Quantification of Msh4 foci number in synaptic forks of spermatocytes of the indicated genotypes. (two mice analyzed for each genotype). Error bars = mean±SD; p= p value.

Article Snippet: Sources and dilutions of antibodies for immunofluorescence were as follows: mouse anti-SYCP3 (Santacruz, sc-74569) 1:300; rabbit anti-SYCP3 (Novus Biologicals nb300-231) 1:300; rabbit anti-SYCP1 (Abcam) 1:200; rabbit anti-DMC1 (Santacruz, sc 22768) 1:100; rabbit anti-γH2AX (Cell signaling) 1:400-500; guinea pig anti-H1T (a gift from M.A.

Techniques: Labeling

Journal: bioRxiv

Article Title: H2AFX and MDC1 Protect Genomic Integrity in Male Germ Cells by Promoting Recombination and Activation of the Recombination-Dependent Checkpoint

doi: 10.1101/235085

Figure Lengend Snippet: (A and C) Quantification of MLH3 foci in 60 dpp mice of the indicated genotypes and stages (three mice analyzed for each genotype). Each dot in the graph indicates the number of MLH3 foci per nucleus; the black bars are means and standard deviation, p= p values (see S1 table for more details). (B) Representative images of chromosome spreads from the indicated stages and genotypes, stained with antibodies against SYCP3 (green) and MLH3 (red). White arrows point MLH3 foci.

Article Snippet: Sources and dilutions of antibodies for immunofluorescence were as follows: mouse anti-SYCP3 (Santacruz, sc-74569) 1:300; rabbit anti-SYCP3 (Novus Biologicals nb300-231) 1:300; rabbit anti-SYCP1 (Abcam) 1:200; rabbit anti-DMC1 (Santacruz, sc 22768) 1:100; rabbit anti-γH2AX (Cell signaling) 1:400-500; guinea pig anti-H1T (a gift from M.A.

Techniques: Standard Deviation, Staining

Journal: bioRxiv

Article Title: H2AFX and MDC1 Protect Genomic Integrity in Male Germ Cells by Promoting Recombination and Activation of the Recombination-Dependent Checkpoint

doi: 10.1101/235085

Figure Lengend Snippet: (A and D) Quantification of DMC1 foci number in adult mice of the indicated genotypes and stages. Sub-classes of spermatocytes were identified by looking at the intensity of H1t staining (namely: no staining= early pachynema, faint staining= early to mid-pachynema, intense staining= mid-pachynema). Each dot in the graphs indicate the number of DMC1 foci per nucleus. Three mice analyzed for each genotype. (B) Representative images of nuclear spreads of indicated genotypes labeled with anti-SYCP3 (red), anti-H1t (white, in the insert) and anti-DMC1 (green) in cells with the indicated genotypes. Magnification bar is 5 μ m. (C and E) Percentage of H1t-positive and H1t-negative spermatocytes from mice of the indicated genotypes. The black bars in A and D are means and SD. p= p values. Error bars in C and E are SD. n= number of analysed cells.

Article Snippet: Sources and dilutions of antibodies for immunofluorescence were as follows: mouse anti-SYCP3 (Santacruz, sc-74569) 1:300; rabbit anti-SYCP3 (Novus Biologicals nb300-231) 1:300; rabbit anti-SYCP1 (Abcam) 1:200; rabbit anti-DMC1 (Santacruz, sc 22768) 1:100; rabbit anti-γH2AX (Cell signaling) 1:400-500; guinea pig anti-H1T (a gift from M.A.

Techniques: Staining, Labeling

Journal: Yonsei Medical Journal

Article Title: Changes in Inward Rectifier K + Channels in Hepatic Stellate Cells During Primary Culture

doi: 10.3349/ymj.2008.49.3.459

Figure Lengend Snippet: Relative inward rectifier K + channel and SUR gene expression in HSC. (A) The inward rectifier K + channel α-subunit gene expression in HSC was measured using real-time RT-PCR. The bar graphs show the relative gene expression for each inward rectifier K + channel subfamily (K ir 1.1, K ir 2.1 - K ir 2.4, K ir 3.1 - K ir 3.4, K ir 4.1 - K ir 4.2, K ir 5.1, K ir 6.1 - K ir 6.2, and K ir 7.1). (B) SUR gene expression was measured. The bar graphs show the relative gene expression for SUR1, SUR2A, and SUR2B. The HSC were used at 1 day, 1 week, 2 weeks, and 3 weeks of culture. The expression levels were normalized to GAPDH and calibrated by K ir 2.1 expression in the HSC cultured for 1 day. The data are shown as the mean ± SEM (n = 3). (C) K ir 2.1 and K ir 6.1 protein expression were measured by Western blotting. There was a band (55 kD) in the anti-K ir 2.1 membrane (Upper) as well as the anti-K ir 6.1 membrane (Lower). Both membranes were re-probed with anti-GAPDH antibody as shown. The data is representative of three independent experiments.

Article Snippet: The anti-K ir 2.1 and anti-K ir 6.1 antibodies were obtained from Alomone Labs (Jerusalem, Israel), the anti-GAPDH antibody was acquired from Novus Biologicals (Littleton, CO, USA), and Western Lightning was supplied by PerkinElmer Life Sciences (Gaithersburg, MD, USA).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Membrane

Journal: International journal of oncology

Article Title: Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma.

doi: 10.3892/ijo.2016.3822

Figure Lengend Snippet: Figure 4. Detection of genome editing at RPN2 gene. (A) Cloning target RPN2 sequence using the Guide-it CRISPR/Cas9 system. A red line under AGG indicates protospacer-adjacent motif. (B) Fluorescence analysis shows that transfection with RPN2-Cas9-GFP plasmid increased GFP-expressing cells in MKN74 and KATO III cell lines. Fluorescence cell imaging were visualized 96 h following transfection. (C) Indel frequency was measured using the T7E1 assay and the percentages are shown at the bottom of each lane. (D) Protein extracted from fluorescence positive cells was analyzed for RPN2 and GAPDH by western blot assay. (E) Sanger sequencing of PCR products around gRNA binding site (red letters) and protospacer adjacent motif (PAM) site (blue letters). Wild-type reference sequences are given on the top.

Article Snippet: Anti-RPN2 Ab was from Aviva Systems and anti-GAPDH Ab was from IMGENEX.

Techniques: Cloning, Sequencing, CRISPR, Fluorescence, Transfection, Plasmid Preparation, Expressing, Imaging, Western Blot, Binding Assay

Journal: International journal of oncology

Article Title: Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma.

doi: 10.3892/ijo.2016.3822

Figure Lengend Snippet: Figure 6. Relationship between RPN2 and p53 in gastric adenocarcinoma. (A) p53 protein stained in brown was detected in the nucleus of primary gastric adenocarcinoma lesions. (B) Evidence for knockdown of RPN2 caused by transfection with siRNA. MKN45 cells were transfected with scrambled siRNA (control); GAPDH was used as loading control. RPN2 siRNA followed by western blot assays using anti-RPN2 antibodies. (C) The knockout of RPN2 expression in MKN45 cell line did not reduce the ability of invasion. Data are expressed as mean values ± SD from triplicate experiments.

Article Snippet: Anti-RPN2 Ab was from Aviva Systems and anti-GAPDH Ab was from IMGENEX.

Techniques: Staining, Knockdown, Transfection, Control, Western Blot, Knock-Out, Expressing