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: Science Advances

Article Title: Functional neuronal circuits promote disease progression in cancer

doi: 10.1126/sciadv.ade4443

Figure Lengend Snippet: ( A ) Tumor growth curve of MOC7 (HPV − ) tumors orthotopically implanted into C57Bl/6 (control) or TRPV1 cre ::DTA fl/WT mice ( n = 10 mice per group). Statistical analysis by multiple Student’s t test. ** P < 0.01; * P < 0.05. ( B ) Photomicrograph of HNSCC ( n = 4 cases) IHC stained for SP (brown). Scale bar, 50 μm. ( C ) En face confocal image of a HNSCC patient sample immunofluorescently stained for SP (red) and β-III tubulin (green). N = 4 tumors analyzed with similar results. Scale bar, 100 μm. ( D ) Ipsilateral TGM ganglion from tracer injected MOC7 (HPV − ) tumor showing colocalization of tracer (red) and SP (green). Scale bar, 50 μm. ( E ) Representative en face confocal images of HNSCC patient sample immunofluorescently stained for NK1R (red) ( n = 4 patient samples). Scale bar, 100 μm. ( F ) Western blot analysis of whole cell lysates from the indicated HNSCC cell lines for NK1R. β-Actin, loading control. Cellular proliferation of the indicated HNSCC ( G ) or HGSOC ( H ) cell lines in response to increasing concentrations of SP for 24 hours or with inclusion of an anti-NK1R antagonist that inhibits this effect [( I ), HNSCC; ( J ), HGSOC]. Statistical test by one-way ANOVA with post hoc Tukey test. * P < 0.05, ** P < 0.01; *** P < 0.001; **** P < 0.0001. OD, optical density.

Article Snippet: The following antibodies were used: anti–phospho-p44/42 MAP kinase (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, catalog no. 9101, 1:1000, RRID:AB_331646), anti-p44/42 Erk1/2 (Cell Signaling Technology, catalog no. 9102, 1:1000, RRID:AB_330744), β-actin (Sigma Life Science, catalog no. A2228, 1:1000), and anti-NK1R (Novus Biologicals, 1:500, catalog no. NB300-119).

Techniques: Staining, Injection, Western Blot

Journal: Science Advances

Article Title: Functional neuronal circuits promote disease progression in cancer

doi: 10.1126/sciadv.ade4443

Figure Lengend Snippet: Western blot analysis for p-ERK and total-ERK for SCC1 (HPV − ) or SCC47 (HPV + ) cells treated with SP alone ( A and C , respectively) or in the presence of the NK1R antagonist ( B and D , respectively) for indicated time points. Densitometric quantification (p-ERK/total ERK) for SCC1 and SCC47 cells treated with SP alone ( E ) or in the presence of NK1R antagonist ( F ). ( G ) Photomicrographs of Transwell migration assays of the indicated cells alone (control), treated with SP (SP), or treated with SP with the NK1R antagonist (SP + antagonist). Scale bar, 500 μm. ( H ) Quantification of migration assay [in (G)], n = 3 biological replicates, n = 4 technical replicates. Statistical test by one-way ANOVA with post hoc Tukey test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following antibodies were used: anti–phospho-p44/42 MAP kinase (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, catalog no. 9101, 1:1000, RRID:AB_331646), anti-p44/42 Erk1/2 (Cell Signaling Technology, catalog no. 9102, 1:1000, RRID:AB_330744), β-actin (Sigma Life Science, catalog no. A2228, 1:1000), and anti-NK1R (Novus Biologicals, 1:500, catalog no. NB300-119).

Techniques: Western Blot, Migration

Journal: JCI Insight

Article Title: Identification and therapeutic rescue of autophagosome and glutamate receptor defects in C9ORF72 and sporadic ALS neurons

doi: 10.1172/jci.insight.127736

Figure Lengend Snippet: (A) Immunofluorescence images showing NR1+ punctae on neurites of iMNs treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. Scale bar: 2 μm. This experiment was repeated 3 times with similar results. (B and C) NR1+ punctae per unit area in control, C9-ALS (B), or sporadic ALS (C) iMNs. Each gray circle represents the number of NR1+ punctae per area unit on a single neurite (1 neurite quantified per iMN). n = 33 (controls and C9-ALS) or 13 (sporadic) iMNs quantified per line per condition from 2 biologically independent iMN conversions of 2 CTRL, 2 C9-ALS, or 6 sporadic ALS lines. Median ± interquartile range. Kruskal-Wallis testing. (D) Number of calcium transients per 30 seconds in control or C9-ALS iMNs treated with 10 nM inactive 3K3A-APC or 3K3A-APC. n = 21 iMNs per line per condition from 3 biologically independent iMN conversions of 3 CTRL and 3 C9-ALS lines. For the C9-ALS plus 3K3A-APC condition, n = 19 iMNs per line. Median ± interquartile range. Kruskal-Wallis testing. (E) Number of calcium transients per 30 seconds in control or sporadic ALS iMNs treated with inactive 3K3A-APC or 3K3A-APC. n = 20 iMNs per line per condition from 3 biologically independent iMN conversions of 3 CTRL and 1 sporadic line. Median ± interquartile range. Kruskal-Wallis testing. (F) Immunoblotting of surface NR1 after surface protein biotinylation in C9-ALS iMNs generated with NGN2, ISL1, and LHX3 and treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. (G) Quantification of NR1 immunoblotting from F. n = 4 biologically independent iMN conversions. Each gray circle represents an individual sample. The ratio of surface to total transferrin receptor was used to normalize for the membrane protein extraction efficiency and TUJ1 was used to normalize for neuron number. (H) Immunoblotting of surface NR1 after surface protein biotinylation in sporadic ALS iMNs (1 patient) generated with NGN2, ISL1, and LHX3 and treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. The full blot for total TUJ1 is shown. (I) Quantification of NR1 immunoblotting from H. n = 4 biologically independent iMN conversions. Each gray circle represents an individual sample. The ratio of surface to total transferrin receptor was used to normalize for the membrane protein extraction efficiency and TUJ1 was used to normalize for neuron number. The day of differentiation stated on each panel indicates the day of differentiation on which the experimental treatment or time course was initiated. TF, transferrin.

Article Snippet: After snap freezing, tissue was sectioned by cryostat at 20-μm thickness and stained with the following primary antibodies: anti-poly(GP) (catalog 24494-1-AP, ProteinTech), anti-poly(GR) (catalog MABN778, Millipore), anti-poly(PR) (catalog 23979-1-AP, ProteinTech), anti-NR1 (catalog NB300118, Novus), and anti-MAP2 (catalog ab5392, Abcam).

Techniques: Immunofluorescence, Western Blot, Generated, Protein Extraction

Journal: JCI Insight

Article Title: Identification and therapeutic rescue of autophagosome and glutamate receptor defects in C9ORF72 and sporadic ALS neurons

doi: 10.1172/jci.insight.127736

Figure Lengend Snippet: (A) Overview of the experimental procedure for testing the ability of 3K3A-APC to reduce DPR levels in the hippocampus of C9-BAC mice. (B–D) The effect of 10 nM inactive 3K3A-APC or 3K3A-APC on the level of poly(GR)+ punctae in the dentate gyrus of C9-BAC mice. Mean ± SD of the number of poly(GR)+ (B), poly(GP)+ (C), and poly(PR)+ (D) punctae per cell; each data point represents a single cell. Cells quantified from 3 mice per condition, 1-way ANOVA with Tukey’s correction for all comparisons. Scale bars: 10 μm. Dotted lines outline cell bodies. Neuronal area was determined by manual outlining in ImageJ on the basis of the staining pattern provided by TUJ1 or MAP2. (E) Overview of the experimental procedure for inducing NMDA injury in the hippocampus and testing the ability of 3K3A-APC to mitigate this injury. (F and G) The effect of 0.2 μg of 3K3A-APC delivered in a volume of 0.3 μL on NMDA-induced hippocampal injury in C9orf72+/+ and C9orf72+/– mice. Mean ± SEM of n = 3 mice per condition, 1-way ANOVA with Tukey’s correction across all comparisons. Red dashed lines outline the injury sites (F). Vehicle control conditions were published in a previous study (4). (H and I) Immunostaining (H) and quantification (I) of NR1 levels in C9orf72+/– mice treated with vehicle or 0.2 μg of 3K3A-APC delivered in a volume of 0.3 μL (n = 3 mice per condition, 72 cells quantified per condition). Each gray data point represents a single cell. Mean ± interquartile range. Mann-Whitney test.

Article Snippet: After snap freezing, tissue was sectioned by cryostat at 20-μm thickness and stained with the following primary antibodies: anti-poly(GP) (catalog 24494-1-AP, ProteinTech), anti-poly(GR) (catalog MABN778, Millipore), anti-poly(PR) (catalog 23979-1-AP, ProteinTech), anti-NR1 (catalog NB300118, Novus), and anti-MAP2 (catalog ab5392, Abcam).

Techniques: Staining, Immunostaining, MANN-WHITNEY

Journal: Journal of Cardiovascular Pharmacology

Article Title: Cardiac N-methyl d-aspartate Receptors as a Pharmacological Target

doi: 10.1097/fjc.0000000000000424

Figure Lengend Snippet: FIGURE 7. Confirmation of the presence of GluN1, GluN2A, GluN2B, and GluN2C proteins in rat heart. Rat brain tissue homogenate was used as a control. Panel A shows the repre- sentative images in which the subunits were detected by immunoblotting. B, Changes in the GluN2B protein abun- dance in the heart with age of the animal. Actin was used as a loading control.

Article Snippet: Staining was performed with the following primary antibodies: mouse monolconal anti-GluN1 antibody, and rabbit polyclonal anti-GluN2C antibodies (NB300-118 and NB300-107; Novus Biologicals), and rabbit polyclonal anti-GluN2A and mouse monoclonal anti-GluN2B antibodies (ab14596 and ab28373; Abcam).

Techniques: Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The synthetic cannabinoid dehydroxylcannabidiol restores the function of a major GABAA receptor isoform in a cell model of hyperekplexia

doi: 10.1074/jbc.ra119.011221

Figure Lengend Snippet: Figure 1. Effects of DH-CBD on mutant GlyRα1 in HEK-293 cells. A, Schematic diagram of patch-clamp recording on HEK-293 cells. B, Representative trace records and average values of IGly activated by 1 mM glycine in HEK-293 cells expressing GABAAR (α1β2γ2) and/or R271Q mutant α1 GlyR (n=9). C, Chemical structure of THC and DH-CBD. D, Representative trace records and average values of IGly activated by 1 mM glycine in HEK-293 cells co-expressing GABAAR (α1β2γ2) and R271Q mutant α1 GlyR with or without 1 μM, 3 μM, and 10 μM DH-CBD pre-incubation (n=7). E, Dose-response curves of IGly in HEK-293 cells co-expressing GABAAR (α1β2γ2) and R271Q mutant α1 GlyR with or without 10 μM DH-CBD pre-incubation. The data were normalized to maximal amplitude (Imax) of the WT GlyR group (n=6). F, EC50 values of IGly induced by increasing glycine concentrations in HEK-293 cells co-expressing GABAAR (α1β2γ2) and R271Q mutant α1 GlyR with or without 10 μM DH-CBD pre-incubation (n=6). Data are represented as mean ± SD. ** P< 0.01, *** P < 0.001 based on unpaired t-tests; ns, not significant (P > 0.05).

Article Snippet: The primary antibodies against GABAAR α1 (1:100, 06-868, Merck), GlyR α1 (1:500, NB300-113, Novus), GAPDH (1:5000, 60004-1-AP, Proteintech) were used for incubation overnight.

Techniques: Mutagenesis, Patch Clamp, Expressing, Incubation

Journal: Journal of Biological Chemistry

Article Title: The synthetic cannabinoid dehydroxylcannabidiol restores the function of a major GABAA receptor isoform in a cell model of hyperekplexia

doi: 10.1074/jbc.ra119.011221

Figure Lengend Snippet: Figure 2. Effects of DH-CBD on GABAAR in HEK-293 cells co-expressing GlyRα1R271Q and GABAAR. A, Representative trace records and average values of IGABA activated by 1 mM GABA in HEK-293 cells co-expressing GABAAR (α1β2γ2) and wild type or R271Q mutant α1 GlyR (n=13). B, Representative trace records and average values of IGABA activated by 1 mM GABA in HEK-293 cells co-expressing GABAAR (α1β2γ2) and R271Q mutant α1 GlyR with or without 1 μM, 3 μM, and 10 μM DH-CBD pre-incubation (n=8). C, D Dose-response curves (C) and EC50 values (D) of IGABA in HEK-293 cells co-expressing GABAAR (α1β2γ2) (n=6) and R271Q mutant α1 GlyR with or without 10 μM DH-CBD pre-incubation (n=7). The data were normalized to Imax of the GABAAR + GlyRα1WT group. E, Representative trace records and average values of IGABA induced by 1 μM, 30 μM, and 1000 μM GABA in HEK-293 cells expressing GABAAR (α1β2γ2) alone (n=7) with or without 10 μM DH-CBD pre-incubation (n=6). Data are represented as mean ± SD. ** P< 0.01, *** P < 0.001 based on unpaired t-tests; ns, not significant (P > 0.05).

Article Snippet: The primary antibodies against GABAAR α1 (1:100, 06-868, Merck), GlyR α1 (1:500, NB300-113, Novus), GAPDH (1:5000, 60004-1-AP, Proteintech) were used for incubation overnight.

Techniques: Expressing, Mutagenesis, Incubation

Journal: Journal of Biological Chemistry

Article Title: The synthetic cannabinoid dehydroxylcannabidiol restores the function of a major GABAA receptor isoform in a cell model of hyperekplexia

doi: 10.1074/jbc.ra119.011221

Figure Lengend Snippet: Figure 3. Effects of DH-CBD on protein interaction between GABAAR and GlyRα1R271Q. A, GlyRα1 protein was purified using GABAAR α1 antibodies in HEK-293 cells co-expressing GABAAR (α1β2γ2) and WT/R271Q mutant α1 GlyR with or without 10 μM DH-CBD pre- incubation. Input represents the same protein immunoblots extracted from cell lysates prior to co-IP. Quantification of WT and R271Q mutant GlyR α1 binding to GABAAR α1 subunits with or without 10 μM DH-CBD pre-incubation (n = 4). The data were normalized to the WT group without DH-CBD pre-incubation. B, Schematic diagram of plasmids structure and FRET. C, Representative trace records and average values of IGly activated by 1 mM glycine in HEK-293 cells expressing GlyRα1R271Q (n=7) or CFP-GlyRα1R271Q alone (n=7) or YFP-GABAAR (α1β2γ2) and GlyRα1R271Q (n=8). D, Representative trace records and average values of IGABA activated by 1 mM GABA in HEK-293 cells expressing GABAAR (n=7) or YFP-GABAAR (α1β2γ2) alone (n=7) or YFP-GABAAR (α1β2γ2) and CFP-GlyRα1R271Q (n=6). E, Representative images and quantification of HEK-293 cells co-expressing the CFP-tagged GlyRα1R271Q and YFP-tagged GABAAR (n=12). These images were collected separately via CFP and YFP channels at 0 min, 30 min, and 60 min after 10 μM DH-CBD pre-incubation. Scale bar: 5 μm. F, Representative images and quantification of HEK-293 cells co-expressing the CFP-tagged GlyRα1WT and YFP- tagged GABAAR (n=9). These images were collected separately via CFP and YFP channels at 0 min, 30 min, and 60 min after 10 μM DH-CBD pre-incubation. Scale bar: 5 μm. Data are represented as mean ± SD. * P< 0.05, ** P < 0.01 based on unpaired t-tests; ns, not significant (P > 0.05).

Article Snippet: The primary antibodies against GABAAR α1 (1:100, 06-868, Merck), GlyR α1 (1:500, NB300-113, Novus), GAPDH (1:5000, 60004-1-AP, Proteintech) were used for incubation overnight.

Techniques: Purification, Expressing, Mutagenesis, Incubation, Western Blot, Co-Immunoprecipitation Assay, Binding Assay