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: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: (A) Model-estimated fold change and associated SE of [ 3 H]CCK-8 accumulation (1 μM, 3 min) in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. control (CTL) in HEK293-OATP1B3 cells without any pre-incubation (Co-incubation). (B) Model-estimated fold change and associated SE of [ 3 H]CCK-8 accumulation (1 μM, 3 min) in HEK293-OATP1B3 cells pretreated with bortezomib (Btz) vs. vehicle CTL at each indicated pretreatment concentration and time (Pre-incubation). After pretreatment, cells were washed three times with the HBSS buffer, and the [ 3 H]CCK-8 accumulation was determined in the absence of bortezomib. (C) Model-estimated fold change and associated SE of [ 3 H]CCK-8 accumulation (1 μM, 3 min) in human SCH pretreated with bortezomib (Btz) (50 and 250 nM, 7 h) vs. vehicle CTL. After pretreatment, cells were washed three times with the HBSS buffer, and the [ 3 H]CCK-8 accumulation was determined in the absence of bortezomib. Model-estimated fold change and associated SE of OATP1B3-mediated [ 3 H]pitavastatin (1 μM, 1 min) (D) and [ 3 H]E 2 17βG accumulation (1 μM, 2 min) (E) in bortezomib pretreatment vs. vehicle CTL at each indicated pretreatment concentration and time (Pre-incubation). In D and E, HEK293-OATP1B3 and HEK293-Mock cells were pretreated with vehicle control (CTL) or bortezomib at the indicated concentrations and time. After washing with the HBSS buffer, OATP1B3-mediated [ 3 H]pitavastatin (D) and [ 3 H]E 2 17βG accumulation (E) was determined by subtracting the values determined in the HEK293-Mock cells from those in HEK293-OATP1B3 cells. (F) Model-estimated fold change and associated SE in [ 3 H]CCK-8 accumulation (1 μM, 3 min) vs. CTL. Cells were pre-incubated with bortezomib-free (CTL) or 50 nM bortezomib-containing media for 2 h. At the end of pre-incubation, the culture medium was removed. After washing, CTL- and bortezomib-pretreated cells were cultured in bortezomib-free medium for the indicated time duration. [ 3 H]CCK-8 (1 μM, 3 min) accumulation was determined at the indicated time points after washing three times ( n = 3 in triplicate). A generalized linear mixed model was fit to the data in A-F as described in the “Materials and Methods” (n = 3 for A, D-F; n = 6 for B; n = 5 for C; all experiments were performed in triplicate). To account for multiple comparisons, p-values were adjusted based on the Bonferroni method. * indicates a statistically significant difference (adjusted p <0.05) vs. CTL.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: CCK-8 Assay, Control, Incubation, Concentration Assay, Cell Culture

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: Human SCH were cultured as described in the Materials and Methods section. (A) Immunoblot of OATP1B3 and OATP1B1 in whole cell lysates of human SCH that were treated with bortezomib (Btz) (50 and 250 nM) or vehicle control (CTL). β-actin served as the loading control. Representative images are shown from n = 3 and 4 donors for 50 and 250 nM treatment, respectively. (B) Fold changes of OATP1B1 and OATP1B3 protein levels. Densitometry of OATP1B1 and OATP1B3 protein levels was normalized to that of β-actin. Fold changes of total protein levels of OATP1B1 and OATP1B3 in bortezomib-treated cells vs. CTL were expressed as mean ± SD n = 3 and 4 donors for 50 and 250 nM treatment, respectively. (C) Immunoblot of ubiquitin in whole cell lysates of human SCH treated with bortezomib (Btz) (50 nM, 7 h) or vehicle control. β-actin served as the loading control. Representative images are shown from n = 3 donors.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Cell Culture, Western Blot, Control, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: HEK293-OATP1B1 cells were seeded at a density of 1.2 x 10 5 cells/well in a 24-well plate and were cultured to confluence. Human SCH were cultured as described in the “Materials and Methods”. (A) Model-estimated fold change and associated SE in [ 3 H]pitavastatin accumulation (1 μM, 0.5 min) in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. vehicle control (CTL) in HEK293-OATP1B1 cells (Co-incubation). (B) Model-estimated fold change and associated SE in [ 3 H]pitavastatin accumulation (1 μM, 0.5 min) in HEK293-OATP1B1 cells pretreated with bortezomib (Btz) vs. vehicle control (CTL) at each indicated time and concentration (Pre-incubation). Following pretreatment, cells were washed three times with HBSS buffer, and the [ 3 H]pitavastatin accumulation was determined in the absence of bortezomib. (C) Model-estimated fold change and associated SE in [ 3 H]E 2 17βG accumulation in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. vehicle CTL in HEK293-OATP1B1 cells (Co-incubation). (D) Model-estimated fold change and associated SE in [ 3 H]E 2 17βG accumulation (1 μM, 2 min) vs. vehicle CTL treatment in HEK293-OATP1B1 cells pretreated with bortezomib (Btz) for the indicated times and at the indicated concentrations (Pre-incubation). Following pretreatment, cells were washed three times with HBSS buffer, and the [ 3 H]E 2 17βG accumulation was determined in the absence of bortezomib. (E) Model-estimated fold change and associated SE of [ 3 H]pitavastatin accumulation (1 μM, 0.5 min) in human SCH pre-incubated with bortezomib (50 nM for 7 h) or co-incubated with positive control bromosulfophthalein (BSP) (100 μM) vs. vehicle CTL. Fold changes and SEs were estimated by linear mixed effects models, as described in the “Data Analysis” section (n = 3 hepatocyte donors in triplicate). To account for multiple comparisons, p-values were adjusted based on the Bonferroni method. * indicates a statistically significant difference (adjusted p <0.05) vs. CTL.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Cell Culture, Control, Incubation, Concentration Assay, Positive Control

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: HEK293-FLAG-OATP1B1 and–FLAG-OATP1B3 stable cell lines were seeded at a density of 3x10 6 cells per 100-mm 2 dish. After culturing for 48 h, cells were treated with bortezomib (Btz) (50 nM, 7 h) or vehicle control (CTL). Whole cell lysates (WCL) (500 μg) from HEK293-FLAG-OATP1B1 (A, left panel) and HEK293-FLAG-OATP1B3 (B, left panel) were subjected to immunoprecipitation (IP) with FLAG antibody, followed by immunoblotting with ubiquitin antibody. Immunoblots of FLAG (A and B, middle panels) and ubiquitin (right panels of A and B) were conducted using whole cell lysates (50 μg) of HEK293-FLAG-OATP1B1 (A) and HEK293-FLAG-OATP1B3 (B) cells treated with bortezomib (50 nM, 7 h) or vehicle control. β-actin served as the loading control. FLAG-OATP1B1 and FLAG-OATP1B3 protein levels determined by densitometry were normalized to levels of β-actin. Fold changes of total protein levels in bortezomib-treated cells vs. CTL were expressed as mean ± SD. Representative images from n = 3 independent experiments are shown.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Stable Transfection, Control, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: HEK293-OATP1B3 cells were seeded at a density of 1.2 x 10 5 cells/well in 24-well plates, and were cultured for 48 h prior to performing the experiment. (A) The concentration-dependent accumulation of CCK-8 (0.1–40 μM, 3 min) was determined in HEK293-OATP1B3 cells pretreated with control (CTL) or bortezomib (Btz) (50 nM, 7 h). Solid and dashed lines represent the best fit lines of the Michaelis–Menten equation to the data of vehicle control (CTL) (closed circles) and bortezomib (Btz) pretreatment (open circles), respectively. A representative graph of three independent experiments performed in triplicate is shown. The student’s t-test was conducted to compare the V max and K m values between bortezomib and vehicle control pretreatment. * indicates a statistically significant difference ( p <0.05; bortezomib vs. CTL). (B) HEK293-OATP1B3 cells were pretreated with 50 nM bortezomib (Btz) or vehicle control (CTL) for 7 h. Surface levels of OATP1B3 were determined via biotinylation, followed by immunoblotting with OATP1B3 and Na-K-ATPase antibodies. GAPDH was used as a cytoplasmic protein marker. OATP1B3 surface protein levels were determined by densitometry and were normalized to those of Na-K-ATPase. Fold changes in the surface levels of OATP1B3 (bortezomib vs. CTL) were expressed as mean ± SD of three independent experiments.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Cell Culture, Concentration Assay, CCK-8 Assay, Control, Western Blot, Marker

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Assessment of Radiolabelled Derivatives of R954 for Detection of Bradykinin B1 Receptor in Cancer Cells: Studies on Glioblastoma Xenografts in Mice.

doi: 10.3390/ph17070902

Figure Lengend Snippet: Figure 2. Specific accumulation of [111In]In-DOTA-Ahx-R954 in cultured U87MG and U251MG cells. (A) Immunoblotting of B1R; (B) Time-dependent changes in cell accumulated radioactivity following

Article Snippet: After blocking with TBST buffer (20 mmol/L Tris–HCl, 150 mmol/L NaCl, 0.5 mL/L Tween 20) containing 5% skim milk, the membranes were incubated with a rabbit polyclonal anti-human BDKRB1 primary antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Cell Culture, Western Blot, Radioactivity

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Assessment of Radiolabelled Derivatives of R954 for Detection of Bradykinin B1 Receptor in Cancer Cells: Studies on Glioblastoma Xenografts in Mice.

doi: 10.3390/ph17070902

Figure Lengend Snippet: Figure 5. B1R expression and intratumoural distribution of GFAP-positive glioblastoma cells. (A) Representative photomicrographs of double immunofluorescent labelling for B1R (green) and

Article Snippet: After blocking with TBST buffer (20 mmol/L Tris–HCl, 150 mmol/L NaCl, 0.5 mL/L Tween 20) containing 5% skim milk, the membranes were incubated with a rabbit polyclonal anti-human BDKRB1 primary antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Expressing

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Assessment of Radiolabelled Derivatives of R954 for Detection of Bradykinin B1 Receptor in Cancer Cells: Studies on Glioblastoma Xenografts in Mice.

doi: 10.3390/ph17070902

Figure Lengend Snippet: Figure 5A shows representative photomicrographs of the U87MG tumour tissue double immunofluorescence labelled for B1R and GFAP, where [111In]In-DOTA-Ahx-R954 showed high accumulation. In this field of view, B1R-positive cells colocalised with GFAP- positive glioblastoma cells. The macro images of GFAP immunoreactivity showed higher fluorescence intensity in the tumour than in the muscle (Figure 5B). Compared to the ex vivo autoradiography of [111In]In-DOTA-Ahx-R954 from the same object shown in Figure 4A, the intratumoural distribution of GFAP-positive cells was consistent with the [111In]In-DOTA-Ahx-R954 accumulated distribution. The tumour-to-muscle ratio of the analysed fluorescence intensity in the images of GFAP-positive cells correlated well with the [111In]In-DOTA-Ahx-R954 accumulation (Figure 5C).

Article Snippet: After blocking with TBST buffer (20 mmol/L Tris–HCl, 150 mmol/L NaCl, 0.5 mL/L Tween 20) containing 5% skim milk, the membranes were incubated with a rabbit polyclonal anti-human BDKRB1 primary antibody (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Immunofluorescence, Fluorescence, Ex Vivo, Autoradiography

Journal: Molecular Pain

Article Title: The effect of kinin B1 receptor on chronic itching sensitization

doi: 10.1186/s12990-015-0070-x

Figure Lengend Snippet: Scratching behaviors in 30 min of DCP-treated mice. Test substances were administered 30 min before observation. Graph showing increased spontaneous scratching of animal model in DCP-treated group ( a ). Preventive treatment with B1R antagonist R892 ( b ), kallikrein inhibitor NFM ( c ) significantly suppressed spontaneous scratching compared with sham-treated group (n = 8), and was significantly different from the vehicle group, while B2R antagonist HOE-140 ( d ) did not. * P < 0.05 as determined by the Student t test

Article Snippet: Paraffin-embedded and formalin-fixed samples were cut into 4-μm sections, IHC staining was performed after dewaxing, 10-min incubation was done with 3 % H 2 O 2 to block endogenous peroxidase, and 25-min microwave antigen was retrieved with B1R antibody (1:500; Biorbyt Ltd, Cambridge, UK) at 4 °C overnight.

Techniques: Animal Model

Journal: Molecular Pain

Article Title: The effect of kinin B1 receptor on chronic itching sensitization

doi: 10.1186/s12990-015-0070-x

Figure Lengend Snippet: B1R is up regulated in skin tissues of DCP-treated group. Western blot ( a ) and RT-PCR ( b ) analysis of B1R in skin tissues obtained from sham (control) and DCP-treated mice. Protein expression ( a ) and PCR product ( b ) obtained with the specific B1R and β-tubulin antibodies or B1R and GAPDH primers. Insets ( a upper panels ) show a representative band of the protein expressed in both groups. Data were normalized against β-tubulin or GAPDH and expressed as the mean ± SEM of five independent mice. ** P < 0.01 by the Student t test

Article Snippet: Paraffin-embedded and formalin-fixed samples were cut into 4-μm sections, IHC staining was performed after dewaxing, 10-min incubation was done with 3 % H 2 O 2 to block endogenous peroxidase, and 25-min microwave antigen was retrieved with B1R antibody (1:500; Biorbyt Ltd, Cambridge, UK) at 4 °C overnight.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Expressing

Journal: Molecular Pain

Article Title: The effect of kinin B1 receptor on chronic itching sensitization

doi: 10.1186/s12990-015-0070-x

Figure Lengend Snippet: Immunofluorescence images of skin tissues in DCP-treated mice. Serial sections of post-fixed skin tissues were used ( a ) showing representative IF images with antibodies to B1R ( green ) and the neuronal marker PGP9.5 ( red ), litter level of B1R was detected in the PGP9.5 positive areas, B1R expression alone ( green ) in keratinocytes or some dermis areas ( b ). A representative IF image with antibodies to PGP9.5 was used to observe morphology of skin

Article Snippet: Paraffin-embedded and formalin-fixed samples were cut into 4-μm sections, IHC staining was performed after dewaxing, 10-min incubation was done with 3 % H 2 O 2 to block endogenous peroxidase, and 25-min microwave antigen was retrieved with B1R antibody (1:500; Biorbyt Ltd, Cambridge, UK) at 4 °C overnight.

Techniques: Immunofluorescence, Marker, Expressing

Journal: Molecular Pain

Article Title: The effect of kinin B1 receptor on chronic itching sensitization

doi: 10.1186/s12990-015-0070-x

Figure Lengend Snippet: HE and IHC in mouse skin tissue. Sections of post-fixed mice skin tissues in DCP and vehicle-treated group immunostained with antibodies to B1R ( a , b ) showing that B1R staining signals were markedly increased in keratinocytes in DCP-treated mice. In photos of both groups, H&E staining was used to observe morphological and pathological changes

Article Snippet: Paraffin-embedded and formalin-fixed samples were cut into 4-μm sections, IHC staining was performed after dewaxing, 10-min incubation was done with 3 % H 2 O 2 to block endogenous peroxidase, and 25-min microwave antigen was retrieved with B1R antibody (1:500; Biorbyt Ltd, Cambridge, UK) at 4 °C overnight.

Techniques: Staining

Journal: Molecular Pain

Article Title: The effect of kinin B1 receptor on chronic itching sensitization

doi: 10.1186/s12990-015-0070-x

Figure Lengend Snippet: B1R expressed in HaCaT cells on a PAR2 background. Positive immunocytochemistry reaction for B1R was observed and displaying membrane and cytosolic localization in HaCaT keratinocytes after 24-h incubation with vehicle (control), PAR2 agonist SLIGKV-NH2 (100 mM) ( a ). Representative images displaying nuclear localization of p65 ( green ) and DAPI ( blue ) in HaCaT keratinocytes after 24-h incubation with vehicle, SLIGKV-NH2 (100 mM) ( c ). Western blot of HaCaT cells following treatment with vehicle (control), SLIGKV-NH2 (100 mM). Samples were probed with antibodies against B1R and tubulin ( b ). B1R mRNA levels were also significantly augmented ( d ). Pretreatment with the NF-κB inhibitor PDTC (25 μM) prevented SLIGKV-NH2-induced B1R expression ( b , d ). Values represent mean ± SEM; * P < 0.05; ** P < 0.01 by the Student t test

Article Snippet: Paraffin-embedded and formalin-fixed samples were cut into 4-μm sections, IHC staining was performed after dewaxing, 10-min incubation was done with 3 % H 2 O 2 to block endogenous peroxidase, and 25-min microwave antigen was retrieved with B1R antibody (1:500; Biorbyt Ltd, Cambridge, UK) at 4 °C overnight.

Techniques: Immunocytochemistry, Membrane, Incubation, Control, Western Blot, Expressing

Journal: iScience

Article Title: Bradykinin induces acute kidney injury after hypothermic circulatory arrest through the repression of the Nrf2-xCT pathway

doi: 10.1016/j.isci.2024.110075

Figure Lengend Snippet: The effect of bradykinin receptor blockade on bradykinin -induced oxidative stress injury and the Nrf2-xCT signaling pathway in vitro and in vivo Representative Western blot images (A) and quantitative analysis of Nrf2 (B) and xCT (C) expression in HK-2 cells treated with the indicated siRNA and then 100 μM H 2 O 2 alone or 100 μM H 2 O 2 plus 100 nM bradykinin for 24 h. Contents of SOD (D) and MDA (E) in HK-2 cells treated with the indicated stimuli. Flow cytometry (F) and statistical analysis (G) of reactive oxygen species (ROS) in HK-2 cells treated with the indicated stimulus. (H) Representative microscopy images of H 2 DCFDA-stained HK-2 cells detected ROS in response to the indicated stimulus. Contents of SOD (I) and MDA (J) in renal tissues harvested 24 h after surgery from the rats in the Sham, HCA, HCA+DALBK, HCA+Ic and HCA+BK groups. Representative Western blot images (K) and quantitative analysis of Nrf2 (L) and xCT (M) expression in renal tissues harvested 24 h after surgery from the rats in the indicated groups. Scale bar: 200 μm (H). The data in Figures 7 B–7E, 7G, 7I, 7G, 7L, and 7M are expressed as the means ± SDs. Statistical significance of Figures 7 B–7D, 7E, 7G, 7I, 7G, 7L, and 7M was determined by ANOVA; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. HCA, hypothermic circulatory arrest; HCA+DALBK, HCA treated with des-Arg9-[Leu8]-BK; HCA+Ic, HCA treated with icatibant; HCA+BK, HCA treated with BK.

Article Snippet: After the HK-2 cells were incubated at 50-70% confluence in 6-well plates for 24 hours, the cells in each well were treated with 100 nM B1R siRNA (Santa Cruz Biotechnology, sc-39878), B2R siRNA (Guangzhou RiboBio) or control siRNA (Santa Cruz Biotechnology, sc-37007).

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Flow Cytometry, Microscopy, Staining

Journal: iScience

Article Title: Bradykinin induces acute kidney injury after hypothermic circulatory arrest through the repression of the Nrf2-xCT pathway

doi: 10.1016/j.isci.2024.110075

Figure Lengend Snippet:

Article Snippet: After the HK-2 cells were incubated at 50-70% confluence in 6-well plates for 24 hours, the cells in each well were treated with 100 nM B1R siRNA (Santa Cruz Biotechnology, sc-39878), B2R siRNA (Guangzhou RiboBio) or control siRNA (Santa Cruz Biotechnology, sc-37007).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software