Journal: Cell Death Discovery

Article Title: Transcriptional landscapes at the intersection of neuronal apoptosis and substance P-induced survival: exploring pathways and drug targets

doi: 10.1038/cddiscovery.2016.50

Figure Lengend Snippet: Pharmacological and transcriptional effects of SP following induction of apoptosis in CGNs. ( a ) Effect of SP and Myc inhibitor on CGNs viability. Primary cultures of CGNs at 6 DIV were switched into serum-free medium containing 5 mM KCl for an acute induction of apoptotic death. After 48 h, neuronal viability was assessed by counting the number of intact nuclei. Neuroprotective effects of 200 nM SP administration was reverted by cotreatment with the Myc inhibitor. Values for neuronal viability represent the mean±S.E.M. of four determinations in two different experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparisons (* P <0.05, ** P <0.01 versus K25; § P <0.01 versus K5; # P <0.01 versus SP treatment). ( b ) Neuroprotective effects of SP were inhibited by the NK1-selective receptor antagonist SR 140333 (25 nM). ( c ) Representative immunofluorescence photomicrographs showing CGNs stained with an antibody against NK1 receptor (red) or NeuN to visualize neurons (green). Total nuclei were stained with Hoechst (blue). ( d ) Genes differentially expressed in control (K25) versus apoptotic (K5) CGNs were defined as ARGs. Genes differentially expressed in apoptotic (K5) CGNs versus those rescued from death by SP treatment (K5+SP) were defined as SRGs. ( e ) Hierarchical clustering. A hierarchical clustering was used to cluster differentially expressed genes in control (K25), apoptotic (K5) and rescued (K5+SP) CGNs. In this two-dimensional presentation, each row represents a single SRG, whereas the columns represent each of the three different experimental conditions analyzed (K25, K5, K5+SP). As shown in the color bar, the red color indicates upregulation and the green color downregulation.

Article Snippet: Cells were then incubated with the primary polyclonal antibody raised against NK1 receptor (1 : 400 in PBS; Abcam, Cambridge, UK) or NeuN (1 : 200; Sigma-Aldrich) overnight at +4 °C, and then washed in PBS and incubated with a goat anti-rabbit rhodamine-conjugated secondary antibody (1 : 1000; Sigma-Aldrich) for 30 min at room temperature.

Techniques: Immunofluorescence, Staining

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in dormant blastocysts via CB1. (A) Localization of CB1 in dormant and activated blastocysts. The trophectoderm cell surface is decorated with CB1. The levels of CB1 are significantly lower in activated blastocysts than those in dormancy. (B) Rapid activation of ERK by anandamide (ANA) in blastocysts. Dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide for the indicated times in minutes. Increased phosphorylation of ERK1/2(p-ERK1/2) and its translocation into nuclei were observed in dormant blastocyst trophectoderm cells within 5 min of their exposure to 7 nM anandamide, reaching a peak between 15 and 30 min. (C) Activation of ERK by anandamide is dose-dependent. Dormant blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide for 15 min. Anandamide at 7 nM activated ERK1/2 in dormant blastocyst trophectoderm cells, whereas it failed to do so at 28 nM. A CB1-selective antagonist SR141716A (SR1) at 7 nM or a MEK1/2 inhibitor U0126 at 1 μM inhibited the activation of ERK1/2 by 7 nM anandamide. (D) Total ERK remained unchanged. No changes in immunointensity for total ERK1/2 were observed in dormant blastocysts exposed to 7 nM anandamide or the vehicle. (E–G) Differential activation of ERK signaling by anandamide in CB mutant dormant blastocysts. CB1–/–, CB2–/–,or CB1–/– × CB2–/– dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide. Activation of ERK1/2 in the presence of 7 nM anandamide for 15 min was abrogated by the CB1 antagonist SR141716A (SR1) in CB2–/– blastocyst, but not in CB1–/– or CB1–/– × CB2–/– blastocysts. Images shown depict TRITC-labeled antigens in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bars, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Translocation Assay, Mutagenesis, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in normal day-4 blastocysts via CB1. Day-4 blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide (ANA) for 15 min. Activation of ERK1/2 at lower (7 nM) but not higher (28 nM) anandamide concentration was observed. A CB1-selective antagonist SR141716A (SR1) inhibited the accumulation of phospho-ERK1/2 (p-ERK1/2) by 7 nM anandamide. Images depict TRITC-labeled antigen in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bar, 20 μm.) Tr, trophectoderm; ICM, inner cell mass.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Concentration Assay, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Cannabinoid agonist CP55,940 induces activation of ERK in differentiating TS cells via CB1. (A) CB1 is expressed in TS cells. This cell line is stably transfected with the GFP gene. Images depict GFP in green, CB1 in red (TS cell surfaces), and the merge in yellow. (Scale bar, 50 μm.) (B and C) Activation of ERK in TS cells by CP55,940 (CP). TS cells were plated and expanded for 48 h. The cells were serum-starved for 5 h then exposed to different concentrations of CP for 15 min or 7 nM CP for the indicated times or to CP at 7 nM in the presence or absence of a MEK1/2 inhibitor (U0126), CB1-selective antagonist SR141716A (SR1), or CB2-selective antagonist SR144528 (SR2) for 5 min. Phosphorylation of ERK1 in differentiating TS cells was rapidly induced by 7 nM CP. U0126 or SR1, but not SR2, inhibited this activation. Quantitative analysis of ERK activation in C is expressed as percentage relative to the maximum band intensity.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Stable Transfection, Transfection

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Inhibition of depolarization-induced Ca2+ influx by 28 nM anandamide in dormant blastocysts. (A) Distribution of Ca2+ channel α subunits, α1B (N-type) and α1C (L-type), in dormant blastocysts. Both inner cell mass (ICM) and trophectoderm cell (Tr) are decorated with α1B and α1C subunits shown in red, Hoechst-labeled nuclei in blue, and the merge in pink. (B) Inhibition of depolarization-induced Ca2+ influx by anandamide (ANA). Ca2+ mobilization in blastocysts was visualized with Fluo-4 acetoxymethyl ester. Depolarization-induced Ca2+ influx in dormant blastocysts after exposure to 60 mM KCl was dramatically inhibited by 28 nM anandamide but not by 7 nM. The CB1-selective antagonist SR141716A (SR1) at equimolar concentration reversed this inhibition, but the CB2-selective antagonist SR144528 (SR2) was ineffective. The relative level of intracellular Ca2+ is indicated by the fluorescent intensity, which is displayed in pseudocolor according to the color bar by using lsmib. (Scale bar, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Inhibition, Labeling, Concentration Assay

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Anandamide at 7 nM confers blastocyst competency to implantation via CB1

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: The D1R–NMDAR interaction bidirectionally regulates the surface distribution and dynamics of D1R and NMDAR. (A) Immunostaining of surface D1R-CFP (green) and GluN1 subunit (red) in hippocampal neurons. The yellow arrow shows overlay. (B) Immunostaining of surface D1R-CFP in control or after D1/5R agonist, TAT-t2, or TAT-[N2A15] application. (Scale bar, 250 nm.) (C) Normalized measures of D1R-CFP clusters intensity in control (n = 32 neuronal fields), D1/5R agonist-treated (n = 24 neuronal fields; *P < 0.05 compared with control), TAT-NSt2–treated (non-sense of TAT-t2, n = 19 neuronal fields), TAT-t2–treated (n = 21 neuronal fields; **P < 0.01 compared with TAT-NSt2), TAT-NSt3–treated (non-sense of TAT-t3, n = 11 neuronal fields), TAT-t3–treated (n = 12 neuronal fields; P > 0.05 compared with TAT-NSt3), TAT-[NS15]–treated (n = 27 neuronal fields; P > 0.05), or TAT-[N2A15]–treated (n = 21 neuronal fields, *P < 0.05 compared with TAT-[NS15]) conditions. (D) Representative trajectories (1,000 frames, 20-Hz acquisition rate) of surface single D1R-CFP (Left) (green) (scale bar, 400 nm) and GluN1-NMDAR (Right) (blue) (scale bar, 300 nm) in the absence and presence of either D1/5R agonist (10 µM, 15 min) or TAT-t2 (10 µM, 15 min). Bold dotted line, perisynaptic area; thin dotted line, PSD area. (E) Plot of the MSD of surface D1R-CFP (Upper) (green) and GluN1-NMDAR (Lower) (blue) versus time in presence of TAT-NS or TAT-t2 peptides (10 µM, 15 min). The SEM is included for each data point (D1R: TAT-NS, n = 986 trajectories, and TAT-t2, n = 1,326; GluN1-NMDAR: TAT-NS, n = 198, and TAT-t2, n = 134). (F and G) Representative surface distributions of single D1R-CFP (green) (F) and GluN1-NMDAR (blue) (G) in the synaptic area (PSD + perisynaptic area) in control, D1/5R agonist, and TAT-t2 conditions. Each dot represents the detection of a single receptor during a frame. Comparisons of the time spent in the synaptic area (dwell time) by single D1R-CFP (control, n = 173 trajectories; D1/5R agonist, n = 142, **P < 0.01; TAT-t2, n = 752, *P < 0.05) (F) and GluN1-NMDAR (control, n = 189 trajectories; D1/5R agonist, n = 157, *P < 0.05; TAT-t2, n = 134, **P < 0.01) (G) and the synaptic fraction of detected single D1R-CFP (control, n = 14 neuronal fields; D1/5R agonist, n = 19, **P < 0.01; D1/5R agonist in the presence of dynasore, n = 47, **P < 0.01; TAT-t2, n = 15, ***P < 0.001) (F), D5R-CFP (n = 16, P > 0.05) (F), and GluN1-NMDAR (control, n = 11; D1/5R agonist, n = 15, *P < 0.05; TAT-t2, n = 14, *P < 0.05) (G). Dyn., dynasore; D1/5 ago., D1/5 receptor agonist SKF-38393.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: D1R activation or D1R/GluN1-NMDAR interaction blockade increases synaptic NMDAR content and favors AMPAR synaptic long-term potentiation. (A) (Left) Excitatory postsynaptic current traces recorded at −70 mV and +40 mV from a representative hippocampal CA1 pyramidal cell, before and 10 min after exposure to D1/5R agonist. (Right) Relative change over time of the AMPA/NMDA ratio at CA1 synapses in the absence or presence of D1/5R agonist (n = 13, *P < 0.05 10 min after agonist) and in the absence or presence of vehicle (n = 7, P > 0.05). (B) Surface imaging of GluN1-SEP in neurons incubated with either TAT-NS or TAT-t2 (10 µM). (Scale bar, 5 µm.) (Right) Average value of GluN1-SEP content in the synaptic area after TAT-NS or TAT-t2 application (n = 8 neurons per group, **P < 0.01). (C) Dendritic fragment of a hippocampal neuron expressing Homer 1c-DsRed (Upper) and GluA1-SEP (Lower). SEP only fluoresces at neutral pH when receptors are inserted at the plasma membrane. Ten minutes after chemical LTP induction (cLTP), the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. (Insets) High magnification of a synaptic GluA1-SEP cluster. (Scale bar, 2 µm.) (D) Comparison of the synaptic GluA1-SEP fluorescence intensity before and after cLTP with prior TAT-NS (n = 198 synapses, *P < 0.05) or TAT-t2 (n = 215 synapses, *P < 0.05) (TAT-NS versus TAT-t2; *P < 0.05) application. (E) Schematic model of the D1R–NMDAR surface interplay in hippocampal neurons. D1Rs are highly diffusive at the neuronal surface and are dynamically retained in clusters in the vicinity of glutamate synapses where they interact with NMDAR. Dopamine release disrupts this interaction and favors the lateral redistribution of both receptors: D1Rs freely explore extrasynaptic areas, whereas NMDARs laterally reach the PSD where they impact on the long-term plasticity of glutamate synapses.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Activation Assay, Imaging, Incubation, Expressing, Fluorescence

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Radiosynthesis of S1PR1-specific radiotracer, [ 18 F]TZ4877; (b) representative sagittal microPET images of [ 18 F]TZ4877 in mice. Comparing with sham mice, the tracer uptake was significantly higher in the infected mice, and the increased uptake of the tracer showed S aureus dose dependent; (c) the tracer uptake in the brain was quantified; time-activity curves showed that the tracer uptake in infected mice was significantly higher than mice without infections; (d) the average tracer uptake in the brain from 30 to 50 min of the PET scan showed a dose-dependent manner. Data represent the mean ± SEM, n = 3 for each group.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Imaging, Activity Assay, Infection

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: Biodistribution (%ID/g, mean ± SEM) of S1PR1-specific [ 18 F]TZ4877 in Balb/c mice ( n = 4).

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Mouse Assay

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: Biodistribution of S1PR1-specific [ 18 F]TZ4877 in sham, infected, and infected with treatments mice ( n = 4).

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Infection, Mouse Assay

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Representative sagittal microPET images of [ 18 F]TZ4877 in the hind limb of mice. The tracer uptake was relatively low in the hind limb muscle with a SUV of ~1.5 in sham mice. Comparing with sham mice, the tracer uptake was significantly higher in the hind limb of infected mice; (b) time-activity curves showed that the tracer uptake in infected mice was significantly higher than sham mice; (c) the average tracer uptake in the hind limb muscle from 30 to 50 min of the PET scan showed a ~39% increase of SUV in infected mice with a P value of 0.0082. Data represent the mean ± SEM, n = 3 for each group.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Imaging, Activity Assay, Infection

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: PET measurements of S1PR1-specific [ 18 F]TZ4877 in S aureus -infected and sham mice.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Infection

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: Immunohistochemistry analysis of S1PR1 in hind limb muscle of sham and S aureus -infected mice. S1PR1 was significantly upregulated in the muscle of infected mice (red arrow) comparing with sham mice (green arrow), scale bar = 100 μ m.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Immunohistochemistry, Infection

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Expression and functional analysis of TRPV1 in BMMs and Osteoclasts. (a) BMMs stained for the macrophage marker CD11b (red) and TRPV1 (green) depict the latter’s expression in these cells, both in the absence (upper panel) and presence (lower panel) of RANKL. (b) Expression of TRPV1 (red) in phalloidin-stained osteoclasts (green, upper panel) is confirmed by a peptide segment against anti-TRPV1 antibody (lower panel) that reduces the specific fluorescence signal intensity of the TRPV1 channel.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Expressing, Functional Assay, Staining, Marker, Fluorescence

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Functional analysis of TRPV1 in BMMs. (a) BMMs were assessed for intracellular Ca 2+ levels upon TRPV1 modulation. Representative intensity profiles of Fluo4-AM intensity at different frames are indicated. (b) Time series graphs of intracellular Fluo4-AM intensities across 200 frames of live imaging. The arrow at the x-axis signifies the time of addition of the respective drugs (20th frame). Gray traces are of individual cells, and the black trace represents the average of 50 cells. (c) Compiled average of different treatments of BMMs, individual cell traces omitted.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Functional Assay, Imaging

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Functional analysis of TRPV1 in BMMs grown on the CMT:HEMA hydrogel. (a) BMMs grown on hydrogels to check for the endogenous levels of Ca 2+ using Fluo4-AM Ca 2+ -sensitive dye. TRPV1 activation elevates the intracellular Ca 2+ levels, as is quantified in (b); n = 100 cells; one-way ANOVA; ns: non-significant, **** p < 0.0001. (c) Correlation representation of the area of cells and per unit area intensity of Fluo4-AM depicts strong positive correlations under basal and TRPV1-activated conditions but not upon inhibition of the channel.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Functional Assay, Activation Assay, Inhibition

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Morphological analysis of BMMs grown on the hydrogel. (a) Representative images of BMMs grown on glass or hydrogel in the presence of RANKL and TRPV1 modulators. Right panels denote marked inset of respective images. Phalloidin intensity (b) and morphometric analyses of BMM’s area (c), perimeter (d), length (e), width (f), and LWR (g). n = 18–51 cells per group; one-way ANOVA; ns: non-significant, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques:

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Differentiation propensities of BMMs into osteoclasts grown on hydrogel. (a) Representative TRAP assay of BMMs grown on the hydrogel in the presence of the TRPV1 activator (RTX) and inhibitor (5′-IRTX) under differentiating conditions (MCSF + RANKL). (b,c) Quantitation of TRAP-positive cells and multinucleated cells in the presence of capsaicin (b) and RTX (c) shows elevated osteoclastogenesis as compared to MCSF and CMT:HEMA control groups. n = 5–10; one-way ANOVA; ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: TRAP Assay, Quantitation Assay

Journal: PLoS ONE

Article Title: The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Schönlein Purpura

doi: 10.1371/journal.pone.0120411

Figure Lengend Snippet: HMVEC-d were pre-incubated with plasma of patients (N = 30) with acute HSP, plasma of healthy controls (N = 30), or culture medium alone for 48 hr, and then the cells were harvested and analyzed for the expression of C3aR (A) and CD88 (B) by flow cytometry. The expression levels were presented as mean fluorescence intensity (MFI).

Article Snippet: Cells were then harvested by trypsin, washed by PBS, and labeled by PE-conjugated mouse IgG anti-human C3aR, IgG anti-human CD88, or IgG isotype control (BD Biosciences) at 4°C for 30 min. Stained cells were assayed by a FACSCalibur using the CellQuest Software (BD Biosciences).

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence