Journal: The Journal of Cell Biology

Article Title: Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

doi: 10.1083/jcb.200905105

Figure Lengend Snippet: CR-1 physically associates with all four Notch receptors. (A–D) Flag-tagged CR-1 (CR-Flag) was cotransfected with HA-tagged Notch1 (N1FL-HA; A), Myc-tagged Notch2 (N2FL-Myc; B), HA-tagged Notch3 (N3FL-HA; C), and V5-tagged Notch4 (N4FL-V5; D) in COS-7 cells. IP and immunoblotting (IB) were performed with the indicated antibodies. cl., cleaved Notch proteins; fl., FL Notch proteins. (E) Nonquantitative RT-PCR for Notch receptor expression in NTERA2/D1 cells. M, markers. (F and G) Interaction between endogenous CR-1 and Notch1/2 in NTERA2/D1 cells. (F) Notch1 IP was performed using anti-Notch1 polyclonal antibodies (C20 and AF5317). (G) CR-1 IP was performed with anti–CR-1 goat polyclonal antibody (α–CR-1). Normal goat or sheep IgGs were used as negative controls. Proteins were detected with the indicated antibodies. (H) Flag-tagged CFC1 (CFC-Flag) was cotransfected with N1FL-HA, and co-IP was performed as described in A–D.

Article Snippet: Human Notch1 and -2 and mouse Notch1 proteins were detected with rat Notch1 mAb (bTan 20; Developmental Studies Hybridoma Bank), rat Notch2 mAb (C651.6DbHN; Developmental Studies Hybridoma Bank), and mouse Notch1 mAb (mN1A; Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Co-Immunoprecipitation Assay

Journal: The Journal of Cell Biology

Article Title: Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

doi: 10.1083/jcb.200905105

Figure Lengend Snippet: Deletion analysis of the CR-1–Notch interaction. (A) CR-1 deletions. (B) Notch1 deletions/chimeras. (C–F) Co-IP was performed using anti-Flag (C), anti-HA (D and E), or anti-Myc (F) affinity beads. Proteins were detected with the indicated antibodies. cl., cleaved Notch proteins; EV, empty vector; fl., FL Notch proteins; IB, immunoblot; S.S., signal sequence.

Article Snippet: Human Notch1 and -2 and mouse Notch1 proteins were detected with rat Notch1 mAb (bTan 20; Developmental Studies Hybridoma Bank), rat Notch2 mAb (C651.6DbHN; Developmental Studies Hybridoma Bank), and mouse Notch1 mAb (mN1A; Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Western Blot, Sequencing

Journal: The Journal of Cell Biology

Article Title: Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

doi: 10.1083/jcb.200905105

Figure Lengend Snippet: Intracellular interaction of CR-1 and Notch1. (A) Cell surface biotinylation assay. Transiently transfected COS-7 cells were treated with N -hydroxysuccinimide–PEG 4 -biotin. Co-IP or sequential co-IP was performed with the indicated antibodies. Each indicated band corresponds as follows: 1, biotinylated CR-1; 2, unbiotinylated and glycosylated CR-1; 3, unbiotinylated and unglycosylated CR-1; 4, carryover of 3× Flag peptides used for Flag elution. (B) Effect of glycosylation on the CR-1–Notch1 interaction. COS-7 cells were treated with vehicle or 10 µg/ml tunicamycin for 16 h after transient transfection. Co-IP assays were performed reciprocally. (C) Intracellular localization of CR-1 and Notch1. GFP-tagged CR-1 and N1FL-HA were visualized by a confocal microscope. (D) Cell surface expression of CR-1 after BFA treatment. Transiently transfected COS-7 cells were treated with the indicated concentrations of BFA for 16 h. Cells were stained with PE-conjugated anti–CR-1 mAb, and FACS analysis was performed. (E) Transiently transfected COS-7 cells were treated with vehicle or 2 µg/ml BFA for 16 h, and the co-IP experiment was performed using the indicated antibodies. cl., cleaved Notch proteins; fl., FL Notch proteins; IB, immunoblot.

Article Snippet: Human Notch1 and -2 and mouse Notch1 proteins were detected with rat Notch1 mAb (bTan 20; Developmental Studies Hybridoma Bank), rat Notch2 mAb (C651.6DbHN; Developmental Studies Hybridoma Bank), and mouse Notch1 mAb (mN1A; Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Cell Surface Biotinylation Assay, Transfection, Co-Immunoprecipitation Assay, Glycoproteomics, Microscopy, Expressing, Staining, Western Blot

Journal: The Journal of Cell Biology

Article Title: Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

doi: 10.1083/jcb.200905105

Figure Lengend Snippet: Sensitization of the Notch signaling pathway by CR-1. (A) TP-1 reporter assay of co-cultured CHO cells with L-WT, L-Dll1, or L–Jagged-1 cells. CHO cells were transiently transfected with empty vector (EV) or WT CR-1 expression vector before co-culture. Cotransfection of FL Notch1 (N1FL) was also performed. Mean ± SD is shown for three independent experiments. *, P < 0.05. (B) Sucrose gradient isolation of lipid rafts in transiently transfected CHO cells. Fractions 4–5 correspond to the lipid raft fractions. Transferrin receptor (TfR) or Cholera toxin B (CTxB) was used as a nonraft or lipid raft marker, respectively. IB, immunoblot. (C) S1 cleavage sites of Notch1. ANK, ankyrin domain; S.S., signal sequence; TM, transmembrane domain. (D and E) Enhancement of S1 cleavage of Notch1 by CR-1 expression. CHO cells transiently transfected with the indicated amount of expression vectors were incubated with 10 µM DAPT for 24 h and analyzed by Western blotting. Mean ± SD of densitometric quantification is shown for three independent transfections (E). (F) Blockade of CR-1–induced Notch processing by a furin inhibitor. Transiently transfected CHO cells were treated with the indicated concentrations of a furin inhibitor, Decanoyl-RVKR-chloromethylketone, for 24 h, and Notch processing was analyzed as described in D. (G) Enhanced cell surface expression of Notch1 by CR-1. CHO cells were transfected with the indicated expression vectors, and the cell surface expression level of Notch1 was assessed by FACS analysis. Arrows indicate the peaks of Notch1-transfected populations.

Article Snippet: Human Notch1 and -2 and mouse Notch1 proteins were detected with rat Notch1 mAb (bTan 20; Developmental Studies Hybridoma Bank), rat Notch2 mAb (C651.6DbHN; Developmental Studies Hybridoma Bank), and mouse Notch1 mAb (mN1A; Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Reporter Assay, Cell Culture, Transfection, Plasmid Preparation, Expressing, Co-Culture Assay, Cotransfection, Isolation, Marker, Western Blot, Sequencing, Incubation

Journal: The Journal of Cell Biology

Article Title: Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

doi: 10.1083/jcb.200905105

Figure Lengend Snippet: Functional interaction between endogenous CR-1 and Notch1 in EC cells. (A–C) Endogenous expression of CR-1 and Notch receptors in F9 WT and Cr −/− cells. (A) Nonquantitative RT-PCR. Samples treated without reverse transcription (RT−) were used as negative controls. (B) Quantitative RT-PCR. Mean ± SD is shown for four independent cultures. *, P < 0.05 compared with F9 WT. (C) Western blot analysis. Empty vector– and N1FL-transfected CHO cells (CHO EV and N1FL) were used as negative and positive controls, respectively. cl., cleaved Notch proteins; fl., FL Notch proteins. (D and E) FACS analysis for the cell surface expression of endogenous Notch1 in F9 cells. Mean ± SD is shown for three independent cultures (E). *, P = 0.021. (F) Effect of Cr-1 knockdown on ligand-induced TP-1 reporter activity in F9 cells. Transiently transfected F9 WT or Cr −/− cells were co-cultured with L-WT, L-Dll1, or L–Jagged-1 cells. Cotransfection of N1FL was also performed. Mean ± SD of relative values of relative luciferase units is shown for four independent experiments. *, P < 0.05. (G and H) siRNA knockdown of CR-1 in NTERA2/D1 cells. Suppression of CR-1 protein by siCR-1_1 and siCR-1_2 was evaluated by Western blotting (G) and FACS analysis (H, left). GFP was used as an indicator of transfection efficiency (>95%; H, right). (I) Effect of CR-1 knockdown on ligand-induced Notch target gene expression in NTERA2/D1 cells. siRNA-transfected NTERA2/D1 cells were co-cultured with L-WT, L–Dll1, or L–Jagged-1 cells. Human Notch target gene expression was assessed by quantitative RT-PCR. Mean ± SD of relative values is shown for four independent experiments. *, P < 0.05.

Article Snippet: Human Notch1 and -2 and mouse Notch1 proteins were detected with rat Notch1 mAb (bTan 20; Developmental Studies Hybridoma Bank), rat Notch2 mAb (C651.6DbHN; Developmental Studies Hybridoma Bank), and mouse Notch1 mAb (mN1A; Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Functional Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Transfection, Knockdown, Activity Assay, Cell Culture, Cotransfection, Luciferase, Targeted Gene Expression

Journal: The Journal of Cell Biology

Article Title: Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

doi: 10.1083/jcb.200905105

Figure Lengend Snippet: Oligonucleotide DNA/RNAs used in this study

Article Snippet: Human Notch1 and -2 and mouse Notch1 proteins were detected with rat Notch1 mAb (bTan 20; Developmental Studies Hybridoma Bank), rat Notch2 mAb (C651.6DbHN; Developmental Studies Hybridoma Bank), and mouse Notch1 mAb (mN1A; Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Mutagenesis

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: Functional endogenous TRPM8 channels are expressed in DRG-derived F11 cells and upregulated after menthol treatment. A, Calcium indicator fluorescence intensity of representative F11 cells in the presence of TRPM8 agonist. False color images of F11 cells loaded with Fluo-4 were imaged before and after 500 μm menthol stimulation, and after 1 μm ionomycin treatment. The peak fluorescence intensity was normalized to ionomycin. Gray arrow indicates the cell corresponding to the trace. Roman numerals indicate depicted frames. B, Western blot of TRPM8 in untransfected HEK293T cells, untransfected F11 cells, and F11 cells transfected with TRPM8-pcDNA3. Blotting with anti-TRPM8 antibody results in a 130 kDa band in both untransfected and transfected F11cells, indicating endogenous TRPM8 expression on lane 2. HEK293T cells were used as negative control. Cells were lysed and calibrated for equal protein loading using GAPDH (40 kDa band) as protein loading control. C, TIRFM on immunocytochemical representative samples from untransfected F11 cells before and after menthol treatment. Detection with TRPM8 antibody shows a low and discrete expression pattern of particles before menthol treatment. Samples fixed after 30 s. Menthol treatment showed a significant increase in the number of particles. Scale bar, 2 μm. D, Biotinylation assay on untransfected F11 cells shows an increase in the presence of TRPM8 channels at the PM after menthol treatment. Cells were calibrated for equal protein loading using Transferrin Receptor (TfR; 95 kDa band) as protein loading control. E, TIRFM on membrane sheet preparations before and after menthol treatment. F11 membrane sheets from untransfected cells were formed by a brief ultrasound pulse and directly fixed and stained for TRPM8 detection. Low expression of particles at the PM is observed in basal conditions (left). Samples fixed after 30 s of menthol treatment show an increase in the particle density at cell surface (right). White line indicates membrane boundaries. Scale bar, 2 μm.

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: Functional Assay, Derivative Assay, Fluorescence, Western Blot, Transfection, Expressing, Negative Control, Cell Surface Biotinylation Assay, Staining

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: Menthol and icilin increase near-PM TRPM8-EGFP (enhanced green fluorescent protein) localization in transfected F11 cells. A, Images representing the overall presence of the channel after 200 frames (addition of all frames) basally and after menthol or icilin treatment. Purple shaded regions represent higher intensity, suggesting a transient increase in TRPM8-containing vesicle residency at PM after agonist treatment. B, Time course of the fluorescence signal at the EFF showing that acute menthol treatments evoke a transitory response. C, D, EFF increased significantly in response to TRPM8 agonists. There is a threefold and fourfold increase of fluorescence detection in the presence of menthol and icilin, respectively. E, The mobility of TRPM8-EGFP-containing vesicles is increased after agonist treatment. Color-coded mobility images reflect variation of a position within a 30 s recording after stimulus. Red shades represent most mobile regions. Blue shades represent less mobile zones. F, Distribution histogram showing the fraction of pixels with the specified mobility. An increase in the relative mobility of TRPM8-containing vesicles is shown after brief agonist treatment. Color bars on top define the regions integrated. G, Population statistics of the integrated relative pixel mobility of TRPM8-EGFP-containing vesicles. Pixels were separated into three groups: static, moderately mobile, and highly mobile. Data are mean ± SEM. *p < 0.05. **p < 0.01. Scale bars, 1 μm.

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: Transfection, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: Differences in subgroups of particles are observed by TIRFM before and after agonist treatment. A, Differences in particle diffusion modes in response to agonists. Different diffusion modes are noted in percentage units. Strict Brownian diffusion and anomalous diffusion were gathered in one single group (simple diffusion). B, Corralled particle lifetime after stimulation. TRPM8-containing vesicles augment their corralled lifetime near PM regions after menthol or icilin treatment. Data are mean ± SEM. *p < 0.05. **p < 0.01.

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: Diffusion-based Assay

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: Diffusion constants of TRPM8-EGFP-containing particles change in response to agonists either in HEK293T or F11 cells

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: Diffusion-based Assay

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: TRPM8 channels recruited to the PM are functional. A, Biotinylation assay on HEK293T cells transfected with TRPM8-pcDNA3 showing a change in the amount of receptor at the cell surface after 1 min 500 μm menthol incubation. Cells were calibrated for equal protein loading using TfR (95 kDa band) as protein loading control. B, Quantification of the Western blot in A shows an evident increase in the presence of TRPM8 channels at the PM after menthol treatment. C, Representative set of current traces (120) recorded in HEK293T cells expressing TRPM8 channels for control (left) and menthol-treated (right) cells. Untreated cells and vehicle-incubated cells showed as control (0.05% ethanol) present no differences. Cells were recorded on whole-cell mode and pulsed 120 times from 0 to 160 mV every 1 s. Blue traces represent mean current. D, Variance analysis of cellular response. Variance was plotted against the mean current and fitted to a parabola function of the form Itotal = iNP0, where “I” corresponds to the unitary current, “N” to the number of channels, and Po to the open probability. There is an increase in the average current after 2 min of menthol treatment (500 μm). This increase is explained by a near threefold increment in the number of active channels at the PM and a 15% increase in the Po, and assumes no significant change in the single-channel conductance. n = 6.

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: Functional Assay, Cell Surface Biotinylation Assay, Transfection, Incubation, Western Blot, Expressing

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: Translocation of vesicles containing TRPM8 are modulated by calcium and channel activity. A, Absence of external calcium does not appear to affect normal particle arrival to PM. After stimulation there is not a significant increase of particles near PM, calculated as EFF. B, Increase of internal calcium levels after thapsigargin exposure increases the number of TRPM8-containing vesicles. C, Treatment with intracellular calcium chelator BAPTA-AM abolishes the menthol stimulated recruitment of vesicles. D, Incubations with the selective blocker BCTC are enough to eliminate the effect of menthol on vesicle recruitment. Data are mean ± SEM. *p < 0.05. **p < 0.01.

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: Translocation Assay, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: TRPM8 internalization and recruitment are affected after menthol. A, Distribution of particles follow a two-state model, where equilibrium constant values during exocytic and endocytic processes vary in the presence of menthol. B, Partial inhibition of endocytosis processes after MβCD incubation. Decay constant values (τ) indicate that menthol stimulates MβCD-dependent exocytosis by ∼6 times (blue circles) in comparison with control conditions (gray circles). Purple shaded regions on the TIRF images represent higher intensity, suggesting an increase in TRPM8-containing vesicles at PM. C, Partial inhibition of exocytosis processes induced by cytoD incubation. Top, Images represent overall presence of particles after cytoD incubation. Purple shaded regions represent higher intensity, suggesting a decrease in the exocytic rate. Comparison of decay constant values (τ) shows that menthol has a near twofold effect on cytoD-dependent vesicle endocytosis. n = 6.

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: Inhibition, Incubation

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: Hop-diffusion and signal amplification model. TRPM8 channels located in the cellular surface are dynamic and relatively constant in number under normal conditions. In response to chemical agonists, TRPM8 channels can generate a localized Ca2+ signal allowing a fast recruitment of TRPM8-containing vesicles. This process facilitates vesicle fusion and stabilization, increasing the time vesicles reside at the membrane. This positive feedback loop may help the cell reach the depolarization threshold to originate a sensitized cellular response or, alternatively, provide fresh nondesensitized channels helping to maintain a steady cellular response.

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: Diffusion-based Assay, Amplification

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: Agonist exposure increases cold sensitivity in CMC fibers and inhibition of vesicle exocytosis with BoNTA leads to increased adaptation of TRPM8-mediated responses. A, Original recording from a temperature-insensitive mechanosensitive C fiber recorded from the saphenous-nerve innervation territory of a C57BL/6 mouse. Instantaneous discharge rates in response to repeated menthol application (50 and 500 μm) and subsequent cooling of the receptive field are shown. Menthol produces de novo, partly reversible and reproducible sensitization to cold (interstimulus interval and drug wash-in 240 s). Bottom, Time course of cold stimulus and temperature thresholds of activation. B, Quantification of the menthol effect on cold sensitization as increase in response magnitude (action potentials (APs) per 60 s cold stimulus; n = 5). Control cold stimulus marked with “C,” “Me1,” and “Me2”: 50 μm menthol application to receptive field; “W”: drug wash-off (p = 0.04, Wilcoxon matched pairs test). C–E, Repeated cold stimulation can increase cold response of menthol-sensitive cold nociceptors. C, D, Original recording from a CMC fiber showing instantaneous discharge rates in response to repeated 60 s cooling pulses (C) and averaged responses of 4 fibers (D). Repeated activation of the fiber by cold increased each subsequent response to cooling. Bottom, Time course of cold stimulus and temperature thresholds of activation. Interstimulus interval 3–4 min. E, Magnitude of consecutive cold responses counted as action potentials (APs) per 60 s cold stimulus. CMC fibers with large cold response (white square, dashed line) desensitize. Gray circles represent individual data points. Black circles represent data points with SEM (mean of 4 fibers). Open white circles represent recording plotted in C. F, G, BoNTA pretreatment prevents menthol-induced sensitization to cooling in most CMC fibers. F, Original traces of CMC fibers recorded from (top) an untreated C57BL6 mouse skin–nerve preparation and (bottom) from a BoNTA-pretreated skin shown as instantaneous discharge rates in response to a 60 s cooling stimulus. Menthol 50 μm was present throughout cold stimuli 2–5. Bottom, Temperature time course. G, Bar chart illustrating average cold responses (amount of action potentials per cold stimulus of 60 s) of CMC fibers from untreated skins (gray, n = 5) and BoNTA-treated skins (green, n = 9). Circles represent individual data points. The majority (6 of 9) of CMC fibers from BoNTA-pretreated skins showed poor cold sensitization after menthol treatment. Interstimulus interval 180–240 s. *p < 0.05 (Mann–Whitney U test).

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: Inhibition, Activation Assay, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

doi: 10.1523/JNEUROSCI.3820-13.2015

Figure Lengend Snippet: Menthol increases adaptation in BoNTA-pretreated monomodal cold receptors similar to coding deficits observed in TRPM8-deficient CC fibers. A, Original traces of CC fibers recorded from an untreated C57BL6 mouse skin–nerve preparation (top) and from a BoNTA-pretreated skin (bottom) shown as instantaneous discharge rates in response to a 60 s cooling stimulus. Menthol 50 μm was present throughout a second cold stimulus, and menthol 500 μm throughout the third stimulus. Bottom, Temperature time course. B, Averaged histogram in bins of 1/s illustrating the effect of menthol 50 μm on the cold response of CC fibers from untreated skins (gray, n = 8 (50 μm) and 7 (500 μm)) and BoNTA-treated skins (green, n = 9 (50 μm) and 8 (500 μm)). After menthol treatment, BoNTA-treated CC fibers show increased adaptation, illustrated in reduced dynamic and static responses. *p < 0.05 (Mann–Whitney U test). C, Bar charts aligning magnitude of cold response from CC fibers of untreated (gray), BoNTA-pretreated (green), and untreated TRPM8-deficient skins (orange). *p < 0.05 (Mann–Whitney U test). D, Original recording from a TRPM8−/− CC fiber (orange) shown as instantaneous discharge rate with static and dynamic coding deficits and rapid adaptation during 60 s cooling. Gray represents C57BL6 CC fiber (from A). Bottom, Temperature time course. E, Averaged histogram in bins of 1 s summarizing the cold responses of CC fibers recorded from C57BL/6 mice and TRPM8-deficient mice in response to cooling from 30°C to 10°C. Gray columns represent C57BL/6 CC fibers (n = 17). Green columns represent TRPM8−/− CC fibers (n = 3).

Article Snippet: TRPM8 was detected with a rabbit polyclonal anti-TRPM8 antibody (1:400, Alomone Labs).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: SARS-CoV-2 Omicron-specific mRNA vaccine induces potent and broad antibody responses in vivo

doi: 10.1101/2022.02.14.480449

Figure Lengend Snippet: A , Illustration of mRNA vaccine construct expressing SARS-CoV-2 WA-1 and Omicron spikes. The spike open reading frame were flanked by 5’ untranslated region (UTR), 3’ UTR and polyA tail. The Omicron mutations (red) and HexaPro mutations (black) were numbered based on WA-1 spike residue number. B , Distribution of Omicron spike mutations (magenta) were displayed in one protomer of spike trimer of which NTD, RBD, hinge region and S2 were colored in purple, blue, green and orange respectively (PDB: 7SBL). The HexaPro mutations in S2 were colored in cyan. C , Schematics illustrating the formulation and biophysical characterization of LNP-mRNA. D , Dynamic light scattering derived histogram depicting the particle radius distribution of Omicron spike LNP-mRNA. E , Omicron LNP-mRNA image collected on transmission electron microscope. F , human ACE2 receptor binding of LNP-mRNA encoding Omicron spike expressed in 293T cells as detected by human ACE2-Fc fusion protein and PE-anti-human Fc antibody on Flow cytometry. G , Immunization and sample collection schedule. Retro-orbital blood were collected prior Omicron LNP-mRNA vaccination on day 0, day 13 and day 21. Five mice (n=5) were intramuscularly injected with 10 μg Omicron LNP-mRNA on day 0 (prime, Omicron x 1) and day 14 (boost, Omicron x 2). The plasma and peripheral blood mononuclear cells (PBMCs) were separated from blood for downstream assays. The slight offset of the labels reflects the fact that each of the blood collections were perform prior to the vaccination injections. H , ELISA titration curves over serial log 10 -transformed dilution points of plasma collected from mice before and after immunization with Omicron LNP-mRNA at defined time points. I , Binding antibody titers of plasma from mice vaccinated with Omicron LNP-mRNA against Omicron spike RBD as quantified by area under curve of log 10 -transformed titration curve (Log 10 AUC) in . Each dot in bar graphs represents value from one mouse. Data on dot-bar plots are shown as mean ± s.e.m. with individual data points in plots. One-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. Statistical significance labels: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Non-significant comparisons are not shown, unless otherwise noted as n.s., not significant.

Article Snippet: The spike expression on cell surface were detected by staining cells with human ACE2–Fc chimera (Sino Biological, 10108-H02HG) in MACS buffer (D-PBS with 2 mM EDTA and 0.5% BSA) for 20 min on ice.

Techniques: Construct, Expressing, Derivative Assay, Transmission Assay, Microscopy, Binding Assay, Flow Cytometry, Injection, Enzyme-linked Immunosorbent Assay, Titration, Transformation Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 Omicron-specific mRNA vaccine induces potent and broad antibody responses in vivo

doi: 10.1101/2022.02.14.480449

Figure Lengend Snippet: A. Functional titration curves of Omicron, Delta and WA-1 pseudoviruses in hACE2+ cells. B. Representative Flow Cytometry plots of infectivity of Omicron, Delta and WA-1 pseudoviruses in hACE2+ cells. C. Quantification of infectivity of Omicron, Delta and WA-1 pseudoviruses in hACE2+ cells. D. Neutralization titration curves of plasma from mice administered with PBS or WA-1 and Omicron LNP-mRNA against Omicron (left), Delta (mid) and WA-1 (right) pseudovirus. Pseudovirus infection rate was calculated from percent of GFP positive cells and was plotted against plasma dilution (log 10 transformed) as titration curve.

Article Snippet: The spike expression on cell surface were detected by staining cells with human ACE2–Fc chimera (Sino Biological, 10108-H02HG) in MACS buffer (D-PBS with 2 mM EDTA and 0.5% BSA) for 20 min on ice.

Techniques: Functional Assay, Titration, Flow Cytometry, Infection, Neutralization, Transformation Assay

Journal: mBio

Article Title: Engineering of CD8 + T cells with an HIV-specific synthetic notch receptor to secrete broadly therapeutic antibodies for combining antiviral humoral and cellular immune responses

doi: 10.1128/mbio.03839-24

Figure Lengend Snippet: Engineered cells with a CD4-17b SNR can sense the Env-presenting cells and virions. ( A ) Schematic design of a CD4-17b-BFP cell. The CD4-17b molecule (Myc-tagged) targeting HIV-1 Env was used as the extracellular structural domain to bind to the Notch core and the cytoplasmic transcription factor Gal4-VP64 to generate the required synNotch receptor. Upon stimulation by the Env antigen, the detached transcription factor enters the nucleus and binds to the upstream activating sequence (UAS) of the regulated element, triggering the expression of BFP. The response element vector also carries a constitutive DsRed reporter for cell population analysis and purification. ( B ) The dual-positive rate of CD4-17b-BFP cells. Positive CD4-17b-BFP cells were measured by detecting both DsRed fluorescence and CD4-17b SNR stained with an anti-Myc antibody through flow cytometry, where the anti-Myc antibody was labeled by a secondary antibody conjugated to Alexa Fluor 488. ( C ) Expression of BFP in CD4-17b-BFP cells in response to 293T-gp160 cells. Left, schematic representation of CD4-17b-BFP cells reacting with the target cells. The immunofluorescence image (right) was taken after 48 h of co-culture of CD4-17b-BFP cells (red) with 293T-gp160 cells (green). An anti-Env 3B3 monoclonal antibody and the secondary antibody conjugated to Alexa Fluor 488 were used to detect 293T-gp160 cells. BFP and DsRed dual-positive cells are indicated by white arrows. The scale bar is 20 µm. ( D ) Flow cytometry analysis of the percentage of BFP-positive cells in the DsRed-positive CD4-17b-BFP cells described in panel C. ( E, F ) Flow cytometry analysis of the expression of BFP in CD4-17b-BFP cells (gated by DsRed + expression) after stimulation with gradient virus particles without ( E ) or with ( F ) the addition of 293T-gp160 cells for 24 h, reported as mean fluorescence intensity (MFI). HIV-1 NL4-3 particles were added at 0.05 µg (only in panel E), 0.25 µg, or 1.25 µg of p24. Mock, no virus or target cells added. In panels E and F, three independent experiments were performed and the error bars depict the standard deviation (SD). **, P < 0.01; ***, P < 0.001 (unpaired Student’s t -test).

Article Snippet: For detection of the expression of cell surface synNotch receptor, transduced cells were incubated with an anti-Myc polyclonal antibody (Proteintech, Rosemont, IL) for 1 h at 4°C, washed three times with PBS, then incubated with a secondary Alexa Fluor Plus 488-goat anti-rabbit IgG (H+L) cross-adsorbed antibody (Invitrogen, Carlsbad, CA) at a 1:500 dilution for 45 min at 4°C, and washed three times with PBS before analysis.

Techniques: Sequencing, Expressing, Plasmid Preparation, Purification, Fluorescence, Staining, Flow Cytometry, Labeling, Immunofluorescence, Co-Culture Assay, Virus, Standard Deviation

Journal: mBio

Article Title: Engineering of CD8 + T cells with an HIV-specific synthetic notch receptor to secrete broadly therapeutic antibodies for combining antiviral humoral and cellular immune responses

doi: 10.1128/mbio.03839-24

Figure Lengend Snippet: Functional anti-HIV-1 bNAb and BiTE can be produced after activation of the Jurkat T cells equipped with the synNotch circuits. ( A ) Schematic design of the response elements of the CD4-17b-Ab synNotch circuits. ( B ) The dual-positive rate of CD4-17b-Ab Jurkat cells, as determined by flow cytometry at 7 days after FACS purification, was analyzed through the detection of both Myc-tagged CD4-17b SNR and DsRed. Jurkat cells were engineered with CD4-17b SNR and the response element encoding for VN, VRC01, or N6-αCD3. UTD, untransduced. ( C ) The Ab expression of the CD4-17b-Ab cells in panel B after being stimulated with 293T-gp160 for 24 h was determined by RT-qPCR probed to VRC01, N6-αCD3, or both genes. Mock, stimulated with 293T cells. ( D ) Time course kinetics of VRC01 IgG secretion from CD4-17b-Ab or UTD Jurkat cells stimulated with 293T-gp160 (Env+, solid lines) or 293T (Env−, dashed lines) cells. ( E ) After 24 h of co-incubation of CD4-17b-Ab cells or UTD cells with pseudoviruses with the Env proteins from multiple HIV-1 strains, changes in the supernatant virus infectivity were detected by TZM-bl cells. ( F ) The p24 viral replication curves were measured with the co-culture supernatants of H9-HXB2 cells and CD4-17b-Ab cells or UTD cells. H9-HXB2 cells are H9 cells infected with HIV-1 HXB2 for 3 days. ( G ) The cell number of each group in panel F was monitored within the co-culture course. ( H, I ) Flow cytometry analysis of CD62L ( H ) and CD25 ( I ) T cell activation markers on CD4-17b-Ab Jurkat cells (gated by DsRed + expression) co-incubated with 293T-gp160 cells for 24 h, reported as mean fluorescence intensity (MFI). In panels C to I, three (C to G) or four ( H and I ) independent experiments were performed and the error bars depict SD. In panels C and E to I, statistical analysis was performed by unpaired Student’s t -test ( C and E ), two-way repeated measures analysis of variance ( F and G ), or unpaired Mann-Whitney U-test ( H and I ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: For detection of the expression of cell surface synNotch receptor, transduced cells were incubated with an anti-Myc polyclonal antibody (Proteintech, Rosemont, IL) for 1 h at 4°C, washed three times with PBS, then incubated with a secondary Alexa Fluor Plus 488-goat anti-rabbit IgG (H+L) cross-adsorbed antibody (Invitrogen, Carlsbad, CA) at a 1:500 dilution for 45 min at 4°C, and washed three times with PBS before analysis.

Techniques: Functional Assay, Produced, Activation Assay, Flow Cytometry, Purification, Expressing, Quantitative RT-PCR, Incubation, Virus, Infection, Co-Culture Assay, Fluorescence, MANN-WHITNEY

Journal: mBio

Article Title: Engineering of CD8 + T cells with an HIV-specific synthetic notch receptor to secrete broadly therapeutic antibodies for combining antiviral humoral and cellular immune responses

doi: 10.1128/mbio.03839-24

Figure Lengend Snippet: CD8 + T cells equipped with the bifunctional synNotch circuit can control viral replication and kill HIV-1 latency-reactivated cells. ( A ) Schematic diagram of the generation of CD8 + T cells equipped with the synNotch circuits. ( B ) The dual-positive rate of CD4-17b-Ab CD8 + T cells was determined by flow cytometry 7 days after FACS purification. ( C ) The p24 viral replication curves were measured with the co-culture supernatants of H9-HXB2 cells and CD4-17b-Ab CD8 + T cells or UTD cells. ( D ) The cell number of each group in panel C was monitored within the co-culture course. ( E ) Establishment of an HIV latent infection model using human primary CD4 + T cells. CD4 + T cells were first activated and expanded with anti-CD3 and anti-CD28 antibodies for 2 days. The cells were then infected with HIV-1 HXB2 and maintained with gradually decreasing concentrations of interleukin-2 (IL-2) for 5 days from day 2 post-infection to establish latency. (F to H) The survival of HIV-1 HXB2 latently-infected CD4 + T cells, without ( F ) or with 24 h of PMA ( G ) or JQ1 ( H ) pre-treatment, was analyzed by flow cytometry before and after 24 h of incubation with engineered or UTD CD8 + T cells. Anti-CD19 SNR-transduced cells severed as a non-targeting (NT) control. CD107a expression on the engineered or UTD CD8 + T cells was analyzed by flow cytometry following stimulation with the target cells (left). The relative percentage of survival cells (right) was calculated as described in the legend of . In panels C, D, and F to H, three ( C and D ) or four (F to H) independent experiments were performed and the error bars depict SD. Statistical analysis was performed by repeated measures analysis of variance ( C and D ) or Mann-Whitney U-test (F to H). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: For detection of the expression of cell surface synNotch receptor, transduced cells were incubated with an anti-Myc polyclonal antibody (Proteintech, Rosemont, IL) for 1 h at 4°C, washed three times with PBS, then incubated with a secondary Alexa Fluor Plus 488-goat anti-rabbit IgG (H+L) cross-adsorbed antibody (Invitrogen, Carlsbad, CA) at a 1:500 dilution for 45 min at 4°C, and washed three times with PBS before analysis.

Techniques: Control, Flow Cytometry, Purification, Co-Culture Assay, Infection, Incubation, Expressing, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: A-C. Saturation binding assay using CHO-K1 cells expressing CMKLR1 (A), GPR1 (B) or CCRL2 (C) that were incubated with increasing concentrations of [ 125 I]-chemerin (total binding, ●). Non-specific binding (◯) was determined in the presence of a 100-fold excess of unlabeled chemerin and specific binding (■) was calculated as the difference. D-F. Competition binding assay using CHO-K1 cells expressing CMKLR1 (D), GPR1 (E) or CCRL2 (F) that were incubated with 0.1 nM [ 125 I]-chemerin as tracers and increasing concentrations of unlabelled chemerin (●) or the nonapeptide chemerin 9 (◯) as competitors. The data were normalized for nonspecific binding (0%) in the presence of 300 nM chemerin, and specific binding in the absence of competitor (100%). The displayed data represent the mean ± S.E.M. of three independent experiments.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Saturation Assay, Expressing, Incubation, Binding Assay

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: Binding parameters of CHO-K1 cells expressing human CMKLR1, GPR1 or CCRL2.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Binding Assay, Expressing

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: Binding parameters of CHO-K1 cells expressing human CMKLR1, GPR1 or CCRL2.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Binding Assay, Expressing

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: A CHO-K1 cells expressing CMKLR1, GPR1 or CCRL2 were incubated with 0.1 nM [ 125 I]-[145–157]-chemerin only (TOTAL) or 0.1 nM [ 125 I]-[145–157]-chemerin in combination with an excess of chemerin or chemerin 9 as competitors. B. CHO-K1 cells expressing CMKLR1 or GPR1 were incubated with 0.1 nM [ 125 I]-[145–157]-chemerin as tracer and increasing concentrations of unlabelled chemerin (●) or chemerin 9 (◯) as competitors. The data were normalized for nonspecific binding (0%) in the presence of 300 nM chemerin, and specific binding in the absence of competitor (100%). The displayed data represent the mean ± S.E.M. of three independent experiments.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Expressing, Incubation, Binding Assay

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: Real-time measurement of BRET signal in HEK293T cells coexpressing G protein biosensors and CMKLR1, GPR1, CCRL2, GPR44, GPR33 or FPR1, following stimulation for 1 minute by 100 nM chemerin. Results are expressed as the difference in BRET signals measured in the presence and absence of chemerin. As controls, cells expressing the α2c adrenergic receptor (G αi/o ) were stimulated with UK14304; cells expressing the AT1 angiotensin receptor (G α11/q ) were stimulated with angiotensin II; cells expressing the β2 adrenergic receptor (G αs ) were stimulated with isoproterenol; cells expressing thromboxane A2 receptor (G α12/13 ) were stimulated with U46619. Data represent the mean ± S.E.M. of three to six independent experiments. Statistical significance was assessed using Tukey's test (*** P < 0.0001).

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: Real-time measurement of BRET signal in HEK293T cells expressing G protein biosensors and CMKLR1, following stimulation for 1 minute with increasing concentrations of chemerin (●) or the chemerin-9 nonapeptide (◯). Results are expressed as the difference in BRET signals measured in the presence and absence of chemerin. Data represent the mean ± S.E.M. of three independent experiments.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: Signaling parameters of CMKLR1 and GPR1.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques:

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: A. Real-time measurement of BRET signal in HEK293T cells expressing either β-arrestin1- R Luc only (✳) or together with CMKLR1-Venus (●), GPR1-Venus (◯) or CCRL2-Venus (△), following stimulation by 100 nM chemerin. B. Real-time measurement of BRET signal in HEK293T cells expressing either β-arrestin2- R Luc only (✳) or together with CMKLR1-Venus (●), GPR1-Venus (◯) or CCRL2-Venus (△), following stimulation by 100 nM chemerin. C-D Localization of β-arrestin in cells coexpressing β-arrestin1-EYFP (C) or β-arrestin2-GFP and CMKLR1 or GPR1, before (NS) and 5 minutes after stimulation with 100 nM chemerin. E-F. Real-time measurement of BRET signal in HEK293T cells expressing β-arrestin1- R luc and CMKLR1-Venus or GPR1-Venus following stimulation with increasing concentrations of chemerin (●) or the chemerin 9 nonapeptide (◯). G-H Real-time measurement of BRET signal in HEK293T cells expressing β-arrestin2- R luc and CMKLR1-Venus or GPR1-Venus following stimulation with increasing concentrations of chemerin (●) or the chemerin 9 nonapeptide. Results of BRET experiments are expressed as the difference in BRET signals measured in the presence and absence of chemerin. Data represent the mean ± S.E.M. of three independent experiments.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: Real-time measurement of BRET signal in HEK293T cells expressing β-arrestin1- R luc ( A - B ) or β-arrestin2- R luc ( C - D ) in combination with CMKLR1-Venus or GPR1-Venus, following stimulation with 100 nM chemerin in the absence (●) or the presence of Pertussis toxin (PTX, ◯). Results of BRET experiments are expressed as the difference in BRET signals measured in the presence and absence of chemerin. Data represent the mean ± S.E.M. of three independent experiments.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: Down-regulation of CMKLR1 and GPR1 A. CHO-K1 cells expressing chemerin receptors were incubated with 100 nM chemerin for various periods of time. Cell surface receptor was detected by flow cytometry using a saturating concentration of antibodies specific for CMKLR1 (●), GPR1 (◯) or CCRL2 (△). Results were normalized for the fluorescence of unstimulated cells (100%) and for background fluorescence (0%). Data represent the mean ± S.E.M. of three independent experiments. B. CHO-K1 cells expressing chemerin receptors were first incubated with 125 I-chemerin at 4°C and washed with binding buffer containing 500 mM NaCl to eliminate the unbound tracer. Then, cells were either left at 4°C or shifted to 28°C to allow receptor internalization. After 90 minutes, cells were acid-washed and the amount of radioactivity remaining associated with the cells was measured. Data represent the mean ± S.E.M. of three independent experiments.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Expressing, Incubation, Cell Surface Receptor Assay, Flow Cytometry, Concentration Assay, Fluorescence, Binding Assay, Radioactivity

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: A. Calcium mobilization was measured in CHO-K1 cells using the aequorin-based functional assay. Cells expressing chemerin receptors were stimulated with increasing concentrations of chemerin and luminescence was recorded for 30 s. The results were normalized for the basal luminescence of the cells in absence of agonist (0%) and the maximal response obtained for each cell line with 10 μM ATP (100%). B. Enlarged panel derived from Fig 9 A showing that GPR1 signal triggered by 3 μM chemerin accounts for about 15% of the CMKLR1 signal. Data represent the mean ± S.E.M. of three independent experiments C. Immunoblot detection of phosphorylated ERK1/2 MAP kinases revealed with anti-phospho ERK1/2 (upper panel). CHO-K1 cells expressing chemerin receptors were stimulated with 300 nM chemerin for various times. Detection of total ERK1/2 by Western blotting was used to ascertain that an equal amount of material was loaded in each lane (lower panel). A typical experiment out of three performed independently is shown.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Functional Assay, Expressing, Derivative Assay, Western Blot

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: Mouse embryonic fibroblasts (MEF) expressing CMKLR1 or GPR1 were stimulated with 300 nM chemerin (wlack bars) or buffer only (white bars) for two minutes and phosphorylation of ERK1/2 estimated by Western blotting. Results are expressed as the ratio between the amounts of phospho-ERK1/2 and total ERK1/2 following quantification. Data represent the mean ± S.E.M. of three independent experiments. MEF cells were derived form β-Arr1 KO and WT1 or β-Arr2 KO and WT2 siblings. Data represent the mean ± S.E.M. of three independent experiments. Statistical significance was assessed using Tukey's test (*** P < 0.0001; ** and ## P< 0.001; * and # P< 0.01).

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Expressing, Phospho-proteomics, Western Blot, Derivative Assay

Journal: PLoS ONE

Article Title: Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

doi: 10.1371/journal.pone.0164179

Figure Lengend Snippet: Binding of chemerin to CMKLR1 leads to the activation of Gi/o proteins and arrestins as well as to calcium mobilization, Erk1/2 phosphorylation and internalization of the chemerin–receptor complex. Binding of chemerin to GPR1 leads mainly to the activation arrestins, although we cannot exclude contribution of G protein to GPR1 signaling. A weak calcium mobilization and Erk1/2 phosphorylation is also detected in response chemerin. GPR1 also internalizes efficiently in response to chemerin. In contrast, CCRL2 binds efficiently chemerin but does not signal nor internalize. The current hypotheses is that CCRL2 might present chemerin C-terminus (in red) to nearby cells expressing functional receptors or play the role of chemerin scavenger.

Article Snippet: Chemerin and antibodies used for the detection of human CMKLR1 and CCRL2 receptors were purchased from R&D Systems.

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Expressing, Functional Assay