Journal: PLoS ONE

Article Title: C1 Esterase Inhibitor Reduces Lower Extremity Ischemia/Reperfusion Injury and Associated Lung Damage

doi: 10.1371/journal.pone.0072059

Figure Lengend Snippet: (A and E) Quantification data from immunofluorescence stainings. (B–D) Representative immunofluorescence images of bradykinin receptor b1 as well as ( F – H ) bradykinin receptor b2 staining in vessels of lung tissue. One-way ANOVA followed by Dunnett's post hoc test for significance vs. NaCl controls was used. Error bars indicate mean ± SD. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: Furthermore, we analyzed fibrin deposition (F0111; Dako, Baar, Switzerland), expression of heparan sulfate (HS; 370255, Amsbio, Abingdon, UK), bradykinin receptor b1 (ABR-011, Alomone Labs, Jerusalem, Israel), bradykinin receptor b2 (ABR-012, Alomone Labs) as well as VE-cadherin (sc-6458, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Immunofluorescence, Staining

Journal: Frontiers in Endocrinology

Article Title: The Bradykinin System Contributes to the Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion Fibroblasts: Implications for Term and Preterm Birth

doi: 10.3389/fendo.2022.873727

Figure Lengend Snippet: Pathway of bradykinin synthesis and the expression of the bradykinin system in human amnion. (A) Pathway of bradykinin synthesis. Italic word in brackets is the gene name of the corresponding protein. UD, undetected. (B) FPKM of gene transcripts related to bradykinin synthesis in the amnion at term with labor (TL, n = 3) and without labor (TNL, n = 3) as revealed by transcriptomic sequencing. (C) Immunohistochemical staining of bradykinin B1 receptor (BDKRB1) and bradykinin B2 receptor (BDKRB2) in human amnion, and comparison of BDKRB1 and BDKRB2 mRNA expression in human amnion fibroblasts and epithelial cells ( n = 3). ae, amnion epithelial cells; af, amnion fibroblasts; scale bar, 50 µm. Statistical analysis was performed with unpaired Student’s t -test. ** p < 0.01 vs. ae.

Article Snippet: The primary antibodies used for Western blotting were as follows: p38 (1:500, Cell Signaling, Danvers, MA, USA, #8690), phosphorylated p38 at Thr180/Tyr182 (1:500, Cell Signaling, #4511), ERK1/2 (1:500, Cell Signaling, #4631), phosphorylated ERK1/2 at Thr202/Tyr204 (1:500, Cell Signaling, #4370), JNK (1:1,000, Cell Signaling, #9252), phosphorylated JNK at Thr183/Tyr185 (1:1,000, Cell Signaling, #9255), BDKRB1 (1:100, Santa Cruz, Santa Cruz, CA, USA, #sc-293196), BDKRB2 (1:500, Alomone, Jerusalem BioPark, Israel, #ABR-012), and PTGS2 (1:1,000, Cell Signaling, #12282).

Techniques: Expressing, Sequencing, Immunohistochemical staining, Staining

Journal: Frontiers in Endocrinology

Article Title: The Bradykinin System Contributes to the Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion Fibroblasts: Implications for Term and Preterm Birth

doi: 10.3389/fendo.2022.873727

Figure Lengend Snippet: Primer sequences used for qRT-PCR.

Article Snippet: The primary antibodies used for Western blotting were as follows: p38 (1:500, Cell Signaling, Danvers, MA, USA, #8690), phosphorylated p38 at Thr180/Tyr182 (1:500, Cell Signaling, #4511), ERK1/2 (1:500, Cell Signaling, #4631), phosphorylated ERK1/2 at Thr202/Tyr204 (1:500, Cell Signaling, #4370), JNK (1:1,000, Cell Signaling, #9252), phosphorylated JNK at Thr183/Tyr185 (1:1,000, Cell Signaling, #9255), BDKRB1 (1:100, Santa Cruz, Santa Cruz, CA, USA, #sc-293196), BDKRB2 (1:500, Alomone, Jerusalem BioPark, Israel, #ABR-012), and PTGS2 (1:1,000, Cell Signaling, #12282).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: The Bradykinin System Contributes to the Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion Fibroblasts: Implications for Term and Preterm Birth

doi: 10.3389/fendo.2022.873727

Figure Lengend Snippet: Bradykinin and its receptor abundance in human amnion at term and preterm birth with labor. (A, B) BDKRB1 and BDKRB2 mRNA abundance in the amnion of term labor (TL, n = 12) and term non-labor (TNL, n = 11) groups. (C, D) BDKRB1 and BDKRB2 mRNA abundance in the amnion of preterm labor (PL, n = 11) and preterm non-labor (PNL, n = 7). (E) Bradykinin abundance in the amnion of TL ( n = 14) and TNL ( n = 13) groups. (F) Bradykinin abundance in the amnion of PL ( n = 11) and PNL ( n = 7) groups. Statistical analysis was performed with the Mann–Whitney U test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. TNL or PNL.

Article Snippet: The primary antibodies used for Western blotting were as follows: p38 (1:500, Cell Signaling, Danvers, MA, USA, #8690), phosphorylated p38 at Thr180/Tyr182 (1:500, Cell Signaling, #4511), ERK1/2 (1:500, Cell Signaling, #4631), phosphorylated ERK1/2 at Thr202/Tyr204 (1:500, Cell Signaling, #4370), JNK (1:1,000, Cell Signaling, #9252), phosphorylated JNK at Thr183/Tyr185 (1:1,000, Cell Signaling, #9255), BDKRB1 (1:100, Santa Cruz, Santa Cruz, CA, USA, #sc-293196), BDKRB2 (1:500, Alomone, Jerusalem BioPark, Israel, #ABR-012), and PTGS2 (1:1,000, Cell Signaling, #12282).

Techniques: MANN-WHITNEY

Journal: Frontiers in Endocrinology

Article Title: The Bradykinin System Contributes to the Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion Fibroblasts: Implications for Term and Preterm Birth

doi: 10.3389/fendo.2022.873727

Figure Lengend Snippet: Induction of PTGS2 expression by BK and DABK was mediated by BDKRB2 and BDKRB1, respectively, in human amnion fibroblasts. (A) BDKRB1 inhibitor ELN-441958 failed to block BK (0.1 μM, 4 h)-induced PTGS2 mRNA ( n = 4) and protein ( n = 3) expression. (B) The BDKRB2 inhibitor icatibant blocked BK (0.1 μM, 4 h)-induced PTGS2 mRNA ( n = 4) and protein ( n = 3) expression. (C) The BDKRB1 inhibitor ELN-441958 blocked DABK (0.1 μM, 4 h)-induced PTGS2 mRNA ( n = 4) and protein ( n = 5) expression. (D) The BDKRB2 inhibitor icatibant failed to block DABK (0.1 μM, 4 h)-induced PTGS2 mRNA ( n = 5) and protein ( n = 6) expression. Statistical analysis was performed with one-way ANOVA test followed by Tukey test. Top panels are the representative immunoblots. * p < 0.05, ** p < 0.01 vs. group without BK/DABK and antagonist treatment. # p < 0.05, ## p < 0.01 vs. BK- or DABK-treated groups.

Article Snippet: The primary antibodies used for Western blotting were as follows: p38 (1:500, Cell Signaling, Danvers, MA, USA, #8690), phosphorylated p38 at Thr180/Tyr182 (1:500, Cell Signaling, #4511), ERK1/2 (1:500, Cell Signaling, #4631), phosphorylated ERK1/2 at Thr202/Tyr204 (1:500, Cell Signaling, #4370), JNK (1:1,000, Cell Signaling, #9252), phosphorylated JNK at Thr183/Tyr185 (1:1,000, Cell Signaling, #9255), BDKRB1 (1:100, Santa Cruz, Santa Cruz, CA, USA, #sc-293196), BDKRB2 (1:500, Alomone, Jerusalem BioPark, Israel, #ABR-012), and PTGS2 (1:1,000, Cell Signaling, #12282).

Techniques: Expressing, Blocking Assay, Western Blot

Journal: Frontiers in Endocrinology

Article Title: The Bradykinin System Contributes to the Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion Fibroblasts: Implications for Term and Preterm Birth

doi: 10.3389/fendo.2022.873727

Figure Lengend Snippet: Induction of BDKRB1 and BDKRB2 expression by lipopolysaccharide (LPS) and serum amyloid A1 (SAA1) in human amnion fibroblasts. (A, B) Concentration-dependent induction of BDKRB1 and BDKRB2 mRNA expression by LPS (0, 1, 10, 50 ng/ml, 24 h) ( n = 4) and SAA1 (0, 10, 50, 100 ng/ml, 24 h) ( n = 5). (C, D) Upregulation of BDKRB1 and BDKRB2 protein levels by LPS (50 ng/ml, 24 h) and SAA1 (50 ng/ml, 24 h) ( n = 5). (E, F) The TRL4 inhibitor CLI-095 (5 µM) blocked LPS ( n = 3)- and SAA1 ( n = 4)-induced BDKRB1 and BDKRB2 mRNA expression. Data are the means ± SEM. Statistical analysis was performed with one-way ANOVA test followed by Tukey test. Top panels are the representative immunoblots. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. 0 ng/ml for (A) and (B) ; vs. control (CTR) for (C) and (D) ; vs. group without LPS/SAA1 and CLI-095 treatment for (E) and (F) # p < 0.05, ## p < 0.01 vs. LPS- or SAA1-treated groups.

Article Snippet: The primary antibodies used for Western blotting were as follows: p38 (1:500, Cell Signaling, Danvers, MA, USA, #8690), phosphorylated p38 at Thr180/Tyr182 (1:500, Cell Signaling, #4511), ERK1/2 (1:500, Cell Signaling, #4631), phosphorylated ERK1/2 at Thr202/Tyr204 (1:500, Cell Signaling, #4370), JNK (1:1,000, Cell Signaling, #9252), phosphorylated JNK at Thr183/Tyr185 (1:1,000, Cell Signaling, #9255), BDKRB1 (1:100, Santa Cruz, Santa Cruz, CA, USA, #sc-293196), BDKRB2 (1:500, Alomone, Jerusalem BioPark, Israel, #ABR-012), and PTGS2 (1:1,000, Cell Signaling, #12282).

Techniques: Expressing, Concentration Assay, Western Blot

Journal: Cells

Article Title: Involvement of Bradykinin Receptor 2 in Nerve Growth Factor Neuroprotective Activity

doi: 10.3390/cells9122651

Figure Lengend Snippet: mRNA expression profiles of BK and its receptors genes after NGF treatment, deprivation, and rescue. Transcript levels of genes encoding BK ( Kng1 ; NM_012696), Bradykinin receptor 2 ( Bdkrb2 ; NM_001270713), and Bradykinin receptor 1 ( Bdkrb1 ; NM_030851) in cortical neurons (CNs) following 48 h NGF treatment (+NGF), induction of apoptosis by 6 h NGF deprivation (−NGF 6 h), and rescue by 6 h NGF replacement (+NGF 6 h). Data represent means (±S.E.M.) from four replicates. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparison (* p < 0.001 vs CTR; § p < 0.05 vs CTR 6 h; # p < 0.05 vs. +NGF replacement (+NGF).

Article Snippet: Protein concentration was measured using a Biorad DC protein assay kit (Bio-Rad, Hercules, CA, USA), and equivalent amounts of protein (30 μg) were separated on 4–12% Bis-Tris NuPage precast gels (Invitrogen), blocked with 5% milk in TBS-Tween for 1 h and then incubated overnight with rabbit anti-B2R antibody (Alomone labs 1:1000) or mouse anti-β-actin (Sigma Aldrich 1:10,000).

Techniques: Expressing

Journal: Cells

Article Title: Involvement of Bradykinin Receptor 2 in Nerve Growth Factor Neuroprotective Activity

doi: 10.3390/cells9122651

Figure Lengend Snippet: Expression of B2R after NGF treatment and deprivation in CNs. At 10 DIV, CNs cultured in Neurobasal + 1% B27 (CTR) were treated for 48 h with NGF (100 ng/mL, +NGF) before being deprived of NGF by anti-NGF antibody treatment and incubated for 24 h (−NGF). B2R protein expression was measured by Western blot analysis. The immunoreactive signals at 45 kDa were quantified and normalized against β-actin and expressed as a percentage of the control (CTR). Data represent means (±S.E.M.) from four independent experiments run in duplicate. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparisons (** p < 0.01 versus CTR; # p < 0.01 versus NGF treatment).

Article Snippet: Protein concentration was measured using a Biorad DC protein assay kit (Bio-Rad, Hercules, CA, USA), and equivalent amounts of protein (30 μg) were separated on 4–12% Bis-Tris NuPage precast gels (Invitrogen), blocked with 5% milk in TBS-Tween for 1 h and then incubated overnight with rabbit anti-B2R antibody (Alomone labs 1:1000) or mouse anti-β-actin (Sigma Aldrich 1:10,000).

Techniques: Expressing, Cell Culture, Incubation, Western Blot

Journal: Cells

Article Title: Involvement of Bradykinin Receptor 2 in Nerve Growth Factor Neuroprotective Activity

doi: 10.3390/cells9122651

Figure Lengend Snippet: Expression of B2R in mixed cortical cultures. Representative immunofluorescence images of cultured CNs stained with antibodies for B2R (red), neurons (MAP2, green), astrocytes (GFAP, green), or microglial cells (Iba1, green) and nuclei (Hoechts, blue) in control conditions (CTR) and after NGF treatment (+NGF). B2R immunoreactivity significantly increased in microglial cell body after NGF treatment. Scale bar: 15 µm.

Article Snippet: Protein concentration was measured using a Biorad DC protein assay kit (Bio-Rad, Hercules, CA, USA), and equivalent amounts of protein (30 μg) were separated on 4–12% Bis-Tris NuPage precast gels (Invitrogen), blocked with 5% milk in TBS-Tween for 1 h and then incubated overnight with rabbit anti-B2R antibody (Alomone labs 1:1000) or mouse anti-β-actin (Sigma Aldrich 1:10,000).

Techniques: Expressing, Immunofluorescence, Cell Culture, Staining

Journal: Cells

Article Title: Involvement of Bradykinin Receptor 2 in Nerve Growth Factor Neuroprotective Activity

doi: 10.3390/cells9122651

Figure Lengend Snippet: Expression of bradykinin receptor 2 (B2R) after NGF treatment in cortical microglial cells. ( a ) Western blot analysis of B2R protein expression in enriched microglial cells cultures treated for 48 h with NGF 100 ng/mL (+NGF), normalized against β-actin, and expressed as a percentage of the control (CTR). Data represent means (±S.E.M.) from four independent experiments run in duplicate. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparisons (* p < 0.05). ( b ) Representative immunofluorescence images of microglial cells stained with antibodies for B2R (red) or microglia (Iba1, green) and nuclei (Hoechts, blue) in control conditions (CTR) and after NGF treatment (+NGF). Scale bar: 15 µm.

Article Snippet: Protein concentration was measured using a Biorad DC protein assay kit (Bio-Rad, Hercules, CA, USA), and equivalent amounts of protein (30 μg) were separated on 4–12% Bis-Tris NuPage precast gels (Invitrogen), blocked with 5% milk in TBS-Tween for 1 h and then incubated overnight with rabbit anti-B2R antibody (Alomone labs 1:1000) or mouse anti-β-actin (Sigma Aldrich 1:10,000).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Cells

Article Title: Involvement of Bradykinin Receptor 2 in Nerve Growth Factor Neuroprotective Activity

doi: 10.3390/cells9122651

Figure Lengend Snippet: Effect of B2R antagonism on CN viability. At 10 DIV, CNs cultured in Neurobasal + 1% B27 (CTR) were treated for 48 h with NGF 100 ng/mL (+NGF) and afterwards deprived of NGF for 24 h by washing (−NGF) or rescued by 24 h NGF replacement (+NGF). Neuronal viability was determined by counting intact nuclei and expressed considering 100 as the number of viable neurons in CTR conditions. ( a ) Treatment with HOE140 (1 µM), a selective B2R antagonist, induced a considerable inhibitory effect on the neuroprotection promoted by NGF replacement (HOE140 +NGF). ( b ) In the same conditions, HOE140 significantly antagonized the neuroprotective activity exerted by RPM-7, a selective B2R agonist (RPM-7 (100 nM). Data represent means (±S.E.M.) from four duplicate experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s test (** p < 0.01 versus +NGF; # p < 0.01 versus −NGF; § p < 0.01 versus −NGF).

Article Snippet: Protein concentration was measured using a Biorad DC protein assay kit (Bio-Rad, Hercules, CA, USA), and equivalent amounts of protein (30 μg) were separated on 4–12% Bis-Tris NuPage precast gels (Invitrogen), blocked with 5% milk in TBS-Tween for 1 h and then incubated overnight with rabbit anti-B2R antibody (Alomone labs 1:1000) or mouse anti-β-actin (Sigma Aldrich 1:10,000).

Techniques: Cell Culture, Activity Assay

Journal: Cells

Article Title: Involvement of Bradykinin Receptor 2 in Nerve Growth Factor Neuroprotective Activity

doi: 10.3390/cells9122651

Figure Lengend Snippet: Overexpression of B2R by NGF treatment in 5xFAD brain extracts. Western blot analysis of B2R protein expression (panel ( a ) bands at 45 kDa; panel ( b ) bands at 70kDa) in brain extracts from wild type mice (CTR) and 5xFAD mice intranasally treated with PBS or NGF normalized against rpS6 and expressed as a percentage of the control (CTR). Data represent means (±S.E.M.) from four independent samples. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparisons. (*** p < 0.001).

Article Snippet: Protein concentration was measured using a Biorad DC protein assay kit (Bio-Rad, Hercules, CA, USA), and equivalent amounts of protein (30 μg) were separated on 4–12% Bis-Tris NuPage precast gels (Invitrogen), blocked with 5% milk in TBS-Tween for 1 h and then incubated overnight with rabbit anti-B2R antibody (Alomone labs 1:1000) or mouse anti-β-actin (Sigma Aldrich 1:10,000).

Techniques: Over Expression, Western Blot, Expressing

Journal: Pharmaceuticals

Article Title: Differential Expression of Kinin Receptors in Human Wet and Dry Age-Related Macular Degeneration Retinae

doi: 10.3390/ph13060130

Figure Lengend Snippet: B1 and B2 receptors (B1R, B2R) and vascular endothelial growth factor A (VEGFA) mRNA expression in human retinae ( A – L ). Microphotographs showing the mRNA labeling for B1R (green; B , F , J ), B2R (red; C , G , K ) and VEGFA (purple; D , H , L ) in the control (CTL), dry age-related macular degeneration (AMD) and wet AMD retinae. Sections are counterstained for 4′,6-diamidino-2-phenylindole (DAPI; blue; A , E , I ), which labels cell nuclei. Note that B1R was partially colocalized with B2R in wet AMD in the outer nuclear layer (ONL), and B2R was also partially colocalized with VEGFA in the ONL, however, there was rare colocalization between B1R and VEGFA. Images were obtained at 40×. Scale bar: 20 μm. ( M – O ) Quantification of the mRNA (mean fluorescence intensity/cell) of B1R, B2R and VEGFA in the different layers of the retina. Data are mean ± SEM of values obtained from five retinae per group and four zones per retina. * p < 0.05 AMD compared with control. CTL: control (O), Dry: dry AMD (□), Wet: wet AMD (△), GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer.

Article Snippet: B2R , Rabbit polyclonal, 1:50, ABR-012, Alomone labs, Jerusalem, Israel.

Techniques: Expressing, Labeling, Fluorescence

Journal: Pharmaceuticals

Article Title: Differential Expression of Kinin Receptors in Human Wet and Dry Age-Related Macular Degeneration Retinae

doi: 10.3390/ph13060130

Figure Lengend Snippet: B2R immunoreactivity in human retinae. ( A ) Microphotographs of immunolocalization of B2R in the human control (CTL), dry and wet AMD retinae. All three groups express the same protein levels of B2R, particularly in the GCL and ONL. Sections are counterstained for 4′,6-diamidino-2-phenylindole (DAPI; blue), which labels cell nuclei. Images were obtained at 40×. Scale bar: 20 μm. ( B ) Semi-quantification of B2R fluorescence intensity on all the surface area of each retina from five dry AMD, five wet AMD and five control retinae. Data represent mean intensity ± SEM after subtraction of the background intensity. CTL: control (O), Dry: dry AMD (□), Wet: wet AMD (△), GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer.

Article Snippet: B2R , Rabbit polyclonal, 1:50, ABR-012, Alomone labs, Jerusalem, Israel.

Techniques: Fluorescence

Journal: Pharmaceuticals

Article Title: Differential Expression of Kinin Receptors in Human Wet and Dry Age-Related Macular Degeneration Retinae

doi: 10.3390/ph13060130

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: B2R , Rabbit polyclonal, 1:50, ABR-012, Alomone labs, Jerusalem, Israel.

Techniques: Concentration Assay

Journal: Pharmaceuticals

Article Title: Differential Expression of Kinin Receptors in Human Wet and Dry Age-Related Macular Degeneration Retinae

doi: 10.3390/ph13060130

Figure Lengend Snippet: Summary of the differences in protein expression between the control, dry and wet AMD retinae.

Article Snippet: B2R , Rabbit polyclonal, 1:50, ABR-012, Alomone labs, Jerusalem, Israel.

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: The NGF R100W Mutation Specifically Impairs Nociception without Affecting Cognitive Performance in a Mouse Model of Hereditary Sensory and Autonomic Neuropathy Type V

doi: 10.1523/JNEUROSCI.0688-19.2019

Figure Lengend Snippet: Analysis of DRG markers, sympathetic neurons and innervation, and DRG transcriptome of NGFR100W/wt mice. A, B, Normal expression of NF200, IB4, and CGRP in DRG neurons in both juveniles and adults (A) and in P5 mice (B). Scale bars, 100 μm. C, No significant difference in SCG cell numbers from NGFR100W/wt and NGFwt/wt mice; Student's two-tailed t test: t = 0.482, p = 0.650; NGFwt/wt, n = 4; NGFR100W/wt, n = 3. Scale bar, 100 μm. D, Normal sympathetic innervation of target internal organs in NGFR100W/wt mice with respect to NGFwt/wt controls; heart, Student's two-tailed t test: t = −0.268, p = 0.796; NGFwt/wt, n = 6; NGFR100W/wt, n = 4; stomach, Student's two-tailed t test: t = 0.216, p = 0.835; NGFwt/wt, n = 5; NGFR100W/wt, n = 4; kidney, Student's two-tailed t test: t = −0.002, p = 0.999; NGFwt/wt, n = 6; NGFR100W/wt, n = 4; spleen, Student's two-tailed t test: t = −0.165, p = 0.874; NGFwt/wt, n = 5; NGFR100W/wt, n = 3. Scale bars, 1 mm. E, Reduced expression of B2R and TRPV1 in DRG neurons of adult mice. Scale bar, 50 μm. B2R, Student's two-tailed t test: t = 6.219, **p = 0.003; NGFwt/wt, n = 3; NGFR100W/wt, n = 3. TRPV1, Student's two-tailed t test: t = 12.455, ***p < 0.001; NGFwt/wt, n = 4; NGFR100W/wt, n = 4. F, Volcano plot for differentially expressed genes in DRGs. The x-axis corresponds to log2FC (Log2FoldChange) differential expression, and the y-axis to −Log(FDR; −Log false discovery rate corrected p value). Red and green regions show significantly upregulated and downregulated genes, respectively. The log2FC and −Log(FDR) thresholds are shown as horizontal (−Log(FDR)<0.05) and vertical (|log2FC|>2.0) dashed lines (see Figure 3-1). G, Pathway analysis of differentially expressed genes in DRGs. The gene–gene network was obtained by StringDB tool (https://string-db.org). Colors indicate genes belonging to the main over-represented pathways and functional categories: immune response and chemokines (red and green), phagocytosis (blue and purple), killer cells mediated cytotoxicity (light blue), and rho GTPase (yellow).

Article Snippet: The antibodies and dilutions used were as follows: 1:500 mouse anti-NF200 (Sigma-Aldrich); 1:200 mouse anti-calcitonin gene-related peptide (CGRP; Rockland); 1:100 Invitrogen isolectin GS-B4-biotin conjugate (Thermo Fisher Scientific); 1:200 rabbit anti-B2R (Alomone Labs); 1:300 mouse anti-TRPV1 (Millipore); 1:200 Dako rabbit anti-PGP 9.5 (Agilent Technologies); and 1:300 rabbit anti-NGF M20 (Santa Cruz Biotechnology).

Techniques: Expressing, Two Tailed Test, Functional Assay

Journal: The Journal of Neuroscience

Article Title: The NGF R100W Mutation Specifically Impairs Nociception without Affecting Cognitive Performance in a Mouse Model of Hereditary Sensory and Autonomic Neuropathy Type V

doi: 10.1523/JNEUROSCI.0688-19.2019

Figure Lengend Snippet: Reduced in vitro and in vivo sensitization capability of NGFR100W. A, Bradykinin-induced SP release in DRG cultures is reduced by NGFR100W cotreatment compared with NGFwt. ANOVA-1 (F(2,16) = 10.501, p < 0.002) followed by Student–Newman–Keuls post hoc test, ***p < 0.001, *p = 0.03; hNGFwt, n = 5; hNGFR100W, n = 6; control, n = 6. B, Downregulation of B2R expression by NGFR100W in DRG cultures stimulated with bradykinin. ANOVA-2 (NGF × bradykinin interaction, F(2,45) = 3.371, p = 0.044) followed by Bonferroni post hoc test, ***p < 0.001; hNGFwt-vehicle, n = 7; hNGFR100W-vehicle, n = 8; control-vehicle, n = 8; hNGFwt-bradykinin, n = 8; hNGFR100W- bradykinin, n = 7; control-bradykinin, n = 8. C, Reduced phosphorylation of TRPV1 by NGFR100W in DRG cultures stimulated with bradykinin. ANOVA-2 (NGF × bradykinin interaction, F(2,47) = 9.346, p < 0.001) followed by Bonferroni post hoc test, ***p < 0.001, **p = 0.008, *p = 0.02; n = 8 for each group. D, Human NGFR100W intraplantar injection causes reduced mechanical sensitization compared with human NGFwt. ANOVA-2 repeated-measures test (treatment × time interaction, F(8,144) = 5.785, p < 0.001) followed by Bonferroni post hoc test, hNGFwt versus saline, ***p < 0.001; hNGFwt versus hNGFR100W, ###p < 0.001, ##p = 0.002, hNGFR100W vs saline, n.s. p = 1.000; saline, n = 10; hNGFwt, n = 11; hNGFR100W, n = 9.

Article Snippet: The antibodies and dilutions used were as follows: 1:500 mouse anti-NF200 (Sigma-Aldrich); 1:200 mouse anti-calcitonin gene-related peptide (CGRP; Rockland); 1:100 Invitrogen isolectin GS-B4-biotin conjugate (Thermo Fisher Scientific); 1:200 rabbit anti-B2R (Alomone Labs); 1:300 mouse anti-TRPV1 (Millipore); 1:200 Dako rabbit anti-PGP 9.5 (Agilent Technologies); and 1:300 rabbit anti-NGF M20 (Santa Cruz Biotechnology).

Techniques: In Vitro, In Vivo, Expressing, Injection