anti cxcr4  (Alomone Labs)


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    Alomone Labs anti cxcr4
    Effects of AMD3100 on prazosin and cyclazosin induced ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured and analyzed as in Fig 3 . Cells were pre- incubated with AMD3100 (10 μM, 15 min), followed by stimulation with vehicle (control, ctrl), prazosin (Praz) or cyclazosin (Cycl, 100 μM each) for 20 min. A. Representative images from Western blot experiments with cells transfected with <t>CXCR4.</t> The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in A. Data are mean ± SE. *: p
    Anti Cxcr4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    anti cxcr4 - by Bioz Stars, 2022-07
    93/100 stars

    Images

    1) Product Images from "Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3"

    Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204041

    Effects of AMD3100 on prazosin and cyclazosin induced ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured and analyzed as in Fig 3 . Cells were pre- incubated with AMD3100 (10 μM, 15 min), followed by stimulation with vehicle (control, ctrl), prazosin (Praz) or cyclazosin (Cycl, 100 μM each) for 20 min. A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in A. Data are mean ± SE. *: p
    Figure Legend Snippet: Effects of AMD3100 on prazosin and cyclazosin induced ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured and analyzed as in Fig 3 . Cells were pre- incubated with AMD3100 (10 μM, 15 min), followed by stimulation with vehicle (control, ctrl), prazosin (Praz) or cyclazosin (Cycl, 100 μM each) for 20 min. A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in A. Data are mean ± SE. *: p

    Techniques Used: Incubation, Western Blot, Transfection, Migration

    Screening of adrenergic receptor antagonists for CXCR4 and ACKR3 agonist activity in PRESTO-Tango β-arrestin recruitment assays. Data are mean ± SE from n = 4 independent experiments (in triplicates). Cells were stimulated with vehicle, 100 μM of individual AR antagonists or with 200 nM of CXCL12. Luminescence signals are expressed as fold of vehicle-treated cells (control, = 1). *: p
    Figure Legend Snippet: Screening of adrenergic receptor antagonists for CXCR4 and ACKR3 agonist activity in PRESTO-Tango β-arrestin recruitment assays. Data are mean ± SE from n = 4 independent experiments (in triplicates). Cells were stimulated with vehicle, 100 μM of individual AR antagonists or with 200 nM of CXCL12. Luminescence signals are expressed as fold of vehicle-treated cells (control, = 1). *: p

    Techniques Used: Activity Assay

    Prazosin and cyclazosin induce ERK1/2 phosphorylation. HEK293 cells transfected with CXCR4 ( A/B ) or ACKR3 ( C/D ) were stimulated with CXCL12 (100 nM), prazosin (100 μM) or cyclazosin (100 μM) for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2 (ERK1/2). A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 3 independent experiments as in A. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min. C. Representative images from Western blot experiments with cells transfected with ACKR3. The migration position of molecular mass standards is indicated. D. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in B. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min.
    Figure Legend Snippet: Prazosin and cyclazosin induce ERK1/2 phosphorylation. HEK293 cells transfected with CXCR4 ( A/B ) or ACKR3 ( C/D ) were stimulated with CXCL12 (100 nM), prazosin (100 μM) or cyclazosin (100 μM) for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2 (ERK1/2). A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 3 independent experiments as in A. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min. C. Representative images from Western blot experiments with cells transfected with ACKR3. The migration position of molecular mass standards is indicated. D. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in B. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min.

    Techniques Used: Transfection, Western Blot, Migration

    Prazosin and cyclazosin induce internalization of CXCR4 and ACKR3 in hVSMCs. A. hVSMC were treated with 100 μM cyclazosin or prazosin at 37°C for 15 or 30 min, stained with anti-CXCR4/Alexa 647-conjugated anti-rabbit and anti-ACKR3/Alexa 488-conjugated anti-mouse and analyzed for receptor expression via flow cytometry. RFU: relative fluorescence units. The horizontal and vertical lines show the gating thresholds for CXCR4 (Alexa 647) and ACKR3 (Alexa 488). B-E . Quantification of CXCR4 ( B/C ) and ACKR3 ( D/E ) cell surface expression by flow cytometry. Cells were stimulated with 100 μM cyclazosin ( B/D ) or prazosin ( C/E ) as indicated. Data are mean ± SE from 4 independent experiments. *: p
    Figure Legend Snippet: Prazosin and cyclazosin induce internalization of CXCR4 and ACKR3 in hVSMCs. A. hVSMC were treated with 100 μM cyclazosin or prazosin at 37°C for 15 or 30 min, stained with anti-CXCR4/Alexa 647-conjugated anti-rabbit and anti-ACKR3/Alexa 488-conjugated anti-mouse and analyzed for receptor expression via flow cytometry. RFU: relative fluorescence units. The horizontal and vertical lines show the gating thresholds for CXCR4 (Alexa 647) and ACKR3 (Alexa 488). B-E . Quantification of CXCR4 ( B/C ) and ACKR3 ( D/E ) cell surface expression by flow cytometry. Cells were stimulated with 100 μM cyclazosin ( B/D ) or prazosin ( C/E ) as indicated. Data are mean ± SE from 4 independent experiments. *: p

    Techniques Used: Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence

    Prazosin and cyclazosin induce chemical shift changes in the NMR spectra of CXCR4 and ACKR3 in membranes. 1 H- 13 C HSQC spectra of reductively methylated CXCR4 ( A-C ) and ACKR3 ( D-F ) membrane preparations were recorded without (black) and with (red) 200 μM prazosin ( A/D ), cyclazosin ( B/E ) or atipamezol ( C/F ). Black arrows indicate significant differences in chemical shifts or broadening (loss) of the signal.
    Figure Legend Snippet: Prazosin and cyclazosin induce chemical shift changes in the NMR spectra of CXCR4 and ACKR3 in membranes. 1 H- 13 C HSQC spectra of reductively methylated CXCR4 ( A-C ) and ACKR3 ( D-F ) membrane preparations were recorded without (black) and with (red) 200 μM prazosin ( A/D ), cyclazosin ( B/E ) or atipamezol ( C/F ). Black arrows indicate significant differences in chemical shifts or broadening (loss) of the signal.

    Techniques Used: Nuclear Magnetic Resonance, Methylation

    CXCL12-inudced ERK1/2 phosphorylation in HEK293 cells is augmented after transfection with CXCR4 and ACKR3. HEK293 cells were transfected with empty vector (left), CXCR4 (center) or ACKR3 (right) and stimulated with 100 nM of CXCL12 for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2. The migration position of molecular mass standards is indicated.
    Figure Legend Snippet: CXCL12-inudced ERK1/2 phosphorylation in HEK293 cells is augmented after transfection with CXCR4 and ACKR3. HEK293 cells were transfected with empty vector (left), CXCR4 (center) or ACKR3 (right) and stimulated with 100 nM of CXCL12 for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2. The migration position of molecular mass standards is indicated.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Migration

    Dose-dependent effects of prazosin and cyclazosin in PRESTO-Tango β-arrestin recruitment assays for CXCR4 and ACKR3. Data are mean ± SE from n = 3 independent experiments (in triplicates). RLU: relative luminescence units. A-C: CXCR4 PRESTO-Tango assays. A. Grey circles: cells treated with cyclazosin. Black circles: cells treated with prazosin. B. Cells were pre-incubated with vehicle (control, grey circles) or AMD3100 (10 μM; black circles) for 15 min, followed by stimulation with cyclazosin. **: p
    Figure Legend Snippet: Dose-dependent effects of prazosin and cyclazosin in PRESTO-Tango β-arrestin recruitment assays for CXCR4 and ACKR3. Data are mean ± SE from n = 3 independent experiments (in triplicates). RLU: relative luminescence units. A-C: CXCR4 PRESTO-Tango assays. A. Grey circles: cells treated with cyclazosin. Black circles: cells treated with prazosin. B. Cells were pre-incubated with vehicle (control, grey circles) or AMD3100 (10 μM; black circles) for 15 min, followed by stimulation with cyclazosin. **: p

    Techniques Used: Incubation

    2) Product Images from "Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells"

    Article Title: Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0253821

    Re-organization of α 1A/B/D -AR heteromers after depletion of CXCR4 or ACKR3 in hVSMCs. Quantification of PLA signals for the detection of receptor-receptor interactions in hVSMCs after incubation with non-targeting (NT) siRNA, CXCR4 siRNA, or ACKR3 siRNA, as in Fig 5 . N = 3 independent experiments with n = 10 images per experiment. *: p
    Figure Legend Snippet: Re-organization of α 1A/B/D -AR heteromers after depletion of CXCR4 or ACKR3 in hVSMCs. Quantification of PLA signals for the detection of receptor-receptor interactions in hVSMCs after incubation with non-targeting (NT) siRNA, CXCR4 siRNA, or ACKR3 siRNA, as in Fig 5 . N = 3 independent experiments with n = 10 images per experiment. *: p

    Techniques Used: Proximity Ligation Assay, Incubation

    Re-organization of heteromers between α 1A/B/D -ARs, CXCR4, and ACKR3 upon depletion of AVPR1A in hVSMCs. Quantification of PLA signals for the detection of receptor-receptor interactions in hVSMCs after incubation with non-targeting (NT) siRNA or AVPR1A siRNA, as in Fig 8 . N = 4 independent experiments with n = 10 images per experiment. ΔPLA signals (%NT): Change in PLA signals in cells incubated with AVPR1A siRNA in % of PLA signals in cells incubated with NT siRNA (= 100%). *: p
    Figure Legend Snippet: Re-organization of heteromers between α 1A/B/D -ARs, CXCR4, and ACKR3 upon depletion of AVPR1A in hVSMCs. Quantification of PLA signals for the detection of receptor-receptor interactions in hVSMCs after incubation with non-targeting (NT) siRNA or AVPR1A siRNA, as in Fig 8 . N = 4 independent experiments with n = 10 images per experiment. ΔPLA signals (%NT): Change in PLA signals in cells incubated with AVPR1A siRNA in % of PLA signals in cells incubated with NT siRNA (= 100%). *: p

    Techniques Used: Proximity Ligation Assay, Incubation

    siRNA knockdown of CXCR4 and ACKR3 in hVSMCs. ( A ) Representative PLA images for the detection of CXCR4 in hVSMC after incubation with non-targeting (NT) or CXCR4 siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. ( B ) Representative PLA images for the detection of ACKR3 in hVSMC after incubation with NT or ACKR3 siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm. ( C ) Quantification of PLA signals for the detection of CXCR4 and ACKR3 after incubation with siRNA, as in A/B. N = 3 independent experiments with n = 10 images per experiment. *: p
    Figure Legend Snippet: siRNA knockdown of CXCR4 and ACKR3 in hVSMCs. ( A ) Representative PLA images for the detection of CXCR4 in hVSMC after incubation with non-targeting (NT) or CXCR4 siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. ( B ) Representative PLA images for the detection of ACKR3 in hVSMC after incubation with NT or ACKR3 siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm. ( C ) Quantification of PLA signals for the detection of CXCR4 and ACKR3 after incubation with siRNA, as in A/B. N = 3 independent experiments with n = 10 images per experiment. *: p

    Techniques Used: Proximity Ligation Assay, Incubation

    Detection of heteromers between α 1A/B/D -ARs, CXCR4, and ACKR3 upon depletion of AVPR1A in hVSMCs. Representative PLA images for the detection of receptor-receptor interactions in hVSMC after incubation with non-targeting (NT) siRNA or AVPR1A siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm.
    Figure Legend Snippet: Detection of heteromers between α 1A/B/D -ARs, CXCR4, and ACKR3 upon depletion of AVPR1A in hVSMCs. Representative PLA images for the detection of receptor-receptor interactions in hVSMC after incubation with non-targeting (NT) siRNA or AVPR1A siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm.

    Techniques Used: Proximity Ligation Assay, Incubation

    Detection of α 1A/B/D -AR heteromers after depletion of CXCR4 or ACKR3 in hVSMCs. Representative PLA images for the detection of α 1A/B/D -AR heteromers in hVSMCs after incubation with non-targeting (NT) siRNA (left), CXCR4 siRNA (center), or ACKR3 siRNA (right). Ctrl: Omission of one primary antibody. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm.
    Figure Legend Snippet: Detection of α 1A/B/D -AR heteromers after depletion of CXCR4 or ACKR3 in hVSMCs. Representative PLA images for the detection of α 1A/B/D -AR heteromers in hVSMCs after incubation with non-targeting (NT) siRNA (left), CXCR4 siRNA (center), or ACKR3 siRNA (right). Ctrl: Omission of one primary antibody. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm.

    Techniques Used: Proximity Ligation Assay, Incubation

    3) Product Images from "α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells"

    Article Title: α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.117.006575

    Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.
    Figure Legend Snippet: Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.

    Techniques Used: Derivative Assay, Proximity Ligation Assay

    Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.
    Figure Legend Snippet: Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.

    Techniques Used: Activation Assay

    α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P
    Figure Legend Snippet: α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P

    Techniques Used: Proximity Ligation Assay, Incubation, Small Interfering RNA

    Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P
    Figure Legend Snippet: Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P

    Techniques Used: Proximity Ligation Assay, Incubation, Small Interfering RNA

    4) Product Images from "α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells"

    Article Title: α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.117.006575

    Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.
    Figure Legend Snippet: Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.

    Techniques Used: Derivative Assay, Proximity Ligation Assay

    Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.
    Figure Legend Snippet: Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.

    Techniques Used: Activation Assay

    α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P
    Figure Legend Snippet: α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P

    Techniques Used: Proximity Ligation Assay, Incubation, Small Interfering RNA

    Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P
    Figure Legend Snippet: Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P

    Techniques Used: Proximity Ligation Assay, Incubation, Small Interfering RNA

    5) Product Images from "α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells"

    Article Title: α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.117.006575

    Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.
    Figure Legend Snippet: Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.

    Techniques Used: Derivative Assay, Proximity Ligation Assay

    Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.
    Figure Legend Snippet: Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.

    Techniques Used: Activation Assay

    α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P
    Figure Legend Snippet: α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P

    Techniques Used: Proximity Ligation Assay, Incubation, Small Interfering RNA

    Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P
    Figure Legend Snippet: Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P

    Techniques Used: Proximity Ligation Assay, Incubation, Small Interfering RNA

    6) Product Images from "α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells"

    Article Title: α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.117.006575

    Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.
    Figure Legend Snippet: Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.

    Techniques Used: Derivative Assay, Proximity Ligation Assay

    Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.
    Figure Legend Snippet: Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.

    Techniques Used: Activation Assay

    α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P
    Figure Legend Snippet: α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P

    Techniques Used: Proximity Ligation Assay, Incubation, Small Interfering RNA

    Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P
    Figure Legend Snippet: Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P

    Techniques Used: Proximity Ligation Assay, Incubation, Small Interfering RNA

    7) Product Images from "Exosomal Expression of CXCR4 Targets Cardioprotective Vesicles to Myocardial Infarction and Improves Outcome after Systemic Administration"

    Article Title: Exosomal Expression of CXCR4 Targets Cardioprotective Vesicles to Myocardial Infarction and Improves Outcome after Systemic Administration

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20030468

    In vivo cardio-protection after I/R. ( a ) Scheme depicting the study protocol. I/R model was induced by ligating the left descending anterior coronary that was then released after 30 min. Exo (2 × 10 11 total particles from a pool of n = 6 patients, in 300 μL PBS) were injected tail vein 3 h after reperfusion. ( b ) Exo CXCR4 but not Exo CTRL significantly improved LVEF at 7days after I/R, compared to PBS control group. At 28 days after I/R, Exo CXCR4 , and to a lesser extent Exo CTRL , significantly improved LVEF compared to control group ( n = 6 rats/group), # p
    Figure Legend Snippet: In vivo cardio-protection after I/R. ( a ) Scheme depicting the study protocol. I/R model was induced by ligating the left descending anterior coronary that was then released after 30 min. Exo (2 × 10 11 total particles from a pool of n = 6 patients, in 300 μL PBS) were injected tail vein 3 h after reperfusion. ( b ) Exo CXCR4 but not Exo CTRL significantly improved LVEF at 7days after I/R, compared to PBS control group. At 28 days after I/R, Exo CXCR4 , and to a lesser extent Exo CTRL , significantly improved LVEF compared to control group ( n = 6 rats/group), # p

    Techniques Used: In Vivo, Injection

    ( a ) Masson’s Trichrome staining for the quantification of infarct size. Exo reduce scar size at 28 days after I/R. The reduction in scar size is more pronounced in Exo CXCR4 compared to Exo CTRL treated group ( n = 5 rats/group PBS, n = 6 rats/group Exo CTRL and Exo CXCR4 ). ( b ) Exo CTRL and Exo CXCR4 . increased blood vessel density. SMA, smooth muscle actin (green); TnI, Troponin I (red); nuclear counterstaining (blue); n = 4 rats/group PBS, n = 6 rats/group Exo CTRL and Exo CXCR4 ). # p
    Figure Legend Snippet: ( a ) Masson’s Trichrome staining for the quantification of infarct size. Exo reduce scar size at 28 days after I/R. The reduction in scar size is more pronounced in Exo CXCR4 compared to Exo CTRL treated group ( n = 5 rats/group PBS, n = 6 rats/group Exo CTRL and Exo CXCR4 ). ( b ) Exo CTRL and Exo CXCR4 . increased blood vessel density. SMA, smooth muscle actin (green); TnI, Troponin I (red); nuclear counterstaining (blue); n = 4 rats/group PBS, n = 6 rats/group Exo CTRL and Exo CXCR4 ). # p

    Techniques Used: Staining

    The hCXCR4 expression. ( a ) Flow cytometer analyses for relative expression of hCXCR4 (CD184) in control CPC (CPC CTRL , purple histogram), and CXCR4 overexpressing CPC (CPC CXCR4 , blue histogram). Quantitative data are percentage of positive cells ( n = 6 patients/group) * p
    Figure Legend Snippet: The hCXCR4 expression. ( a ) Flow cytometer analyses for relative expression of hCXCR4 (CD184) in control CPC (CPC CTRL , purple histogram), and CXCR4 overexpressing CPC (CPC CXCR4 , blue histogram). Quantitative data are percentage of positive cells ( n = 6 patients/group) * p

    Techniques Used: Expressing, Flow Cytometry

    CXCR4 expression. ( a ) Quantitative data flow cytometer analyses for relative expression of CXCR4 in Exo CTRL (purple bar), and CXCR4 overexpressing Exo (Exo CXCR4 , blue bar). Exo were bound to beads before analyses as described in the method section. Data are fold-changes in mean fluorescence intensity (MFI) versus controls ( n = 7 patients/group) * p
    Figure Legend Snippet: CXCR4 expression. ( a ) Quantitative data flow cytometer analyses for relative expression of CXCR4 in Exo CTRL (purple bar), and CXCR4 overexpressing Exo (Exo CXCR4 , blue bar). Exo were bound to beads before analyses as described in the method section. Data are fold-changes in mean fluorescence intensity (MFI) versus controls ( n = 7 patients/group) * p

    Techniques Used: Expressing, Flow Cytometry, Fluorescence

    ( a ) Western blot showing pro-survival ERK1/2 phosphorylation in HL-1 CM when Exo CTRL , and Exo CXCR4 were added to the medium in stress-conditions ( n = 4 patients/group) # p
    Figure Legend Snippet: ( a ) Western blot showing pro-survival ERK1/2 phosphorylation in HL-1 CM when Exo CTRL , and Exo CXCR4 were added to the medium in stress-conditions ( n = 4 patients/group) # p

    Techniques Used: Western Blot

    In vitro bioactivities of Exo CXCR4 . ( a , b ) Exo CTRL , and Exo CXCR4 inhibit staurosporine-induced death in HL-1 CM (green) ( n = 6 patients/group for Exo CTRL and Exo CXCR4 ; n = 5 repeated measurements for PBS and FBS) # p
    Figure Legend Snippet: In vitro bioactivities of Exo CXCR4 . ( a , b ) Exo CTRL , and Exo CXCR4 inhibit staurosporine-induced death in HL-1 CM (green) ( n = 6 patients/group for Exo CTRL and Exo CXCR4 ; n = 5 repeated measurements for PBS and FBS) # p

    Techniques Used: In Vitro

    8) Product Images from "Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3"

    Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204041

    Screening of adrenergic receptor antagonists for CXCR4 and ACKR3 agonist activity in PRESTO-Tango β-arrestin recruitment assays. Data are mean ± SE from n = 4 independent experiments (in triplicates). Cells were stimulated with vehicle, 100 μM of individual AR antagonists or with 200 nM of CXCL12. Luminescence signals are expressed as fold of vehicle-treated cells (control, = 1). *: p
    Figure Legend Snippet: Screening of adrenergic receptor antagonists for CXCR4 and ACKR3 agonist activity in PRESTO-Tango β-arrestin recruitment assays. Data are mean ± SE from n = 4 independent experiments (in triplicates). Cells were stimulated with vehicle, 100 μM of individual AR antagonists or with 200 nM of CXCL12. Luminescence signals are expressed as fold of vehicle-treated cells (control, = 1). *: p

    Techniques Used: Activity Assay

    Prazosin and cyclazosin induce ERK1/2 phosphorylation. HEK293 cells transfected with CXCR4 ( A/B ) or ACKR3 ( C/D ) were stimulated with CXCL12 (100 nM), prazosin (100 μM) or cyclazosin (100 μM) for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2 (ERK1/2). A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 3 independent experiments as in A. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min. C. Representative images from Western blot experiments with cells transfected with ACKR3. The migration position of molecular mass standards is indicated. D. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in B. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min.
    Figure Legend Snippet: Prazosin and cyclazosin induce ERK1/2 phosphorylation. HEK293 cells transfected with CXCR4 ( A/B ) or ACKR3 ( C/D ) were stimulated with CXCL12 (100 nM), prazosin (100 μM) or cyclazosin (100 μM) for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2 (ERK1/2). A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 3 independent experiments as in A. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min. C. Representative images from Western blot experiments with cells transfected with ACKR3. The migration position of molecular mass standards is indicated. D. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in B. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min.

    Techniques Used: Transfection, Western Blot, Migration

    Prazosin and cyclazosin induce internalization of CXCR4 and ACKR3 in hVSMCs. A. hVSMC were treated with 100 μM cyclazosin or prazosin at 37°C for 15 or 30 min, stained with anti-CXCR4/Alexa 647-conjugated anti-rabbit and anti-ACKR3/Alexa 488-conjugated anti-mouse and analyzed for receptor expression via flow cytometry. RFU: relative fluorescence units. The horizontal and vertical lines show the gating thresholds for CXCR4 (Alexa 647) and ACKR3 (Alexa 488). B-E . Quantification of CXCR4 ( B/C ) and ACKR3 ( D/E ) cell surface expression by flow cytometry. Cells were stimulated with 100 μM cyclazosin ( B/D ) or prazosin ( C/E ) as indicated. Data are mean ± SE from 4 independent experiments. *: p
    Figure Legend Snippet: Prazosin and cyclazosin induce internalization of CXCR4 and ACKR3 in hVSMCs. A. hVSMC were treated with 100 μM cyclazosin or prazosin at 37°C for 15 or 30 min, stained with anti-CXCR4/Alexa 647-conjugated anti-rabbit and anti-ACKR3/Alexa 488-conjugated anti-mouse and analyzed for receptor expression via flow cytometry. RFU: relative fluorescence units. The horizontal and vertical lines show the gating thresholds for CXCR4 (Alexa 647) and ACKR3 (Alexa 488). B-E . Quantification of CXCR4 ( B/C ) and ACKR3 ( D/E ) cell surface expression by flow cytometry. Cells were stimulated with 100 μM cyclazosin ( B/D ) or prazosin ( C/E ) as indicated. Data are mean ± SE from 4 independent experiments. *: p

    Techniques Used: Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence

    Prazosin and cyclazosin induce chemical shift changes in the NMR spectra of CXCR4 and ACKR3 in membranes. 1 H- 13 C HSQC spectra of reductively methylated CXCR4 ( A-C ) and ACKR3 ( D-F ) membrane preparations were recorded without (black) and with (red) 200 μM prazosin ( A/D ), cyclazosin ( B/E ) or atipamezol ( C/F ). Black arrows indicate significant differences in chemical shifts or broadening (loss) of the signal.
    Figure Legend Snippet: Prazosin and cyclazosin induce chemical shift changes in the NMR spectra of CXCR4 and ACKR3 in membranes. 1 H- 13 C HSQC spectra of reductively methylated CXCR4 ( A-C ) and ACKR3 ( D-F ) membrane preparations were recorded without (black) and with (red) 200 μM prazosin ( A/D ), cyclazosin ( B/E ) or atipamezol ( C/F ). Black arrows indicate significant differences in chemical shifts or broadening (loss) of the signal.

    Techniques Used: Nuclear Magnetic Resonance, Methylation

    CXCL12-inudced ERK1/2 phosphorylation in HEK293 cells is augmented after transfection with CXCR4 and ACKR3. HEK293 cells were transfected with empty vector (left), CXCR4 (center) or ACKR3 (right) and stimulated with 100 nM of CXCL12 for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2. The migration position of molecular mass standards is indicated.
    Figure Legend Snippet: CXCL12-inudced ERK1/2 phosphorylation in HEK293 cells is augmented after transfection with CXCR4 and ACKR3. HEK293 cells were transfected with empty vector (left), CXCR4 (center) or ACKR3 (right) and stimulated with 100 nM of CXCL12 for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2. The migration position of molecular mass standards is indicated.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Migration

    Dose-dependent effects of prazosin and cyclazosin in PRESTO-Tango β-arrestin recruitment assays for CXCR4 and ACKR3. Data are mean ± SE from n = 3 independent experiments (in triplicates). RLU: relative luminescence units. A-C: CXCR4 PRESTO-Tango assays. A. Grey circles: cells treated with cyclazosin. Black circles: cells treated with prazosin. B. Cells were pre-incubated with vehicle (control, grey circles) or AMD3100 (10 μM; black circles) for 15 min, followed by stimulation with cyclazosin. **: p
    Figure Legend Snippet: Dose-dependent effects of prazosin and cyclazosin in PRESTO-Tango β-arrestin recruitment assays for CXCR4 and ACKR3. Data are mean ± SE from n = 3 independent experiments (in triplicates). RLU: relative luminescence units. A-C: CXCR4 PRESTO-Tango assays. A. Grey circles: cells treated with cyclazosin. Black circles: cells treated with prazosin. B. Cells were pre-incubated with vehicle (control, grey circles) or AMD3100 (10 μM; black circles) for 15 min, followed by stimulation with cyclazosin. **: p

    Techniques Used: Incubation

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    Alomone Labs anti cxcr4
    Effects of AMD3100 on prazosin and cyclazosin induced ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured and analyzed as in Fig 3 . Cells were pre- incubated with AMD3100 (10 μM, 15 min), followed by stimulation with vehicle (control, ctrl), prazosin (Praz) or cyclazosin (Cycl, 100 μM each) for 20 min. A. Representative images from Western blot experiments with cells transfected with <t>CXCR4.</t> The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in A. Data are mean ± SE. *: p
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    Effects of AMD3100 on prazosin and cyclazosin induced ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured and analyzed as in Fig 3 . Cells were pre- incubated with AMD3100 (10 μM, 15 min), followed by stimulation with vehicle (control, ctrl), prazosin (Praz) or cyclazosin (Cycl, 100 μM each) for 20 min. A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in A. Data are mean ± SE. *: p

    Journal: PLoS ONE

    Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

    doi: 10.1371/journal.pone.0204041

    Figure Lengend Snippet: Effects of AMD3100 on prazosin and cyclazosin induced ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured and analyzed as in Fig 3 . Cells were pre- incubated with AMD3100 (10 μM, 15 min), followed by stimulation with vehicle (control, ctrl), prazosin (Praz) or cyclazosin (Cycl, 100 μM each) for 20 min. A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in A. Data are mean ± SE. *: p

    Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

    Techniques: Incubation, Western Blot, Transfection, Migration

    Screening of adrenergic receptor antagonists for CXCR4 and ACKR3 agonist activity in PRESTO-Tango β-arrestin recruitment assays. Data are mean ± SE from n = 4 independent experiments (in triplicates). Cells were stimulated with vehicle, 100 μM of individual AR antagonists or with 200 nM of CXCL12. Luminescence signals are expressed as fold of vehicle-treated cells (control, = 1). *: p

    Journal: PLoS ONE

    Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

    doi: 10.1371/journal.pone.0204041

    Figure Lengend Snippet: Screening of adrenergic receptor antagonists for CXCR4 and ACKR3 agonist activity in PRESTO-Tango β-arrestin recruitment assays. Data are mean ± SE from n = 4 independent experiments (in triplicates). Cells were stimulated with vehicle, 100 μM of individual AR antagonists or with 200 nM of CXCL12. Luminescence signals are expressed as fold of vehicle-treated cells (control, = 1). *: p

    Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

    Techniques: Activity Assay

    Prazosin and cyclazosin induce ERK1/2 phosphorylation. HEK293 cells transfected with CXCR4 ( A/B ) or ACKR3 ( C/D ) were stimulated with CXCL12 (100 nM), prazosin (100 μM) or cyclazosin (100 μM) for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2 (ERK1/2). A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 3 independent experiments as in A. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min. C. Representative images from Western blot experiments with cells transfected with ACKR3. The migration position of molecular mass standards is indicated. D. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in B. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min.

    Journal: PLoS ONE

    Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

    doi: 10.1371/journal.pone.0204041

    Figure Lengend Snippet: Prazosin and cyclazosin induce ERK1/2 phosphorylation. HEK293 cells transfected with CXCR4 ( A/B ) or ACKR3 ( C/D ) were stimulated with CXCL12 (100 nM), prazosin (100 μM) or cyclazosin (100 μM) for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2 (ERK1/2). A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 3 independent experiments as in A. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min. C. Representative images from Western blot experiments with cells transfected with ACKR3. The migration position of molecular mass standards is indicated. D. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in B. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min.

    Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

    Techniques: Transfection, Western Blot, Migration

    Prazosin and cyclazosin induce internalization of CXCR4 and ACKR3 in hVSMCs. A. hVSMC were treated with 100 μM cyclazosin or prazosin at 37°C for 15 or 30 min, stained with anti-CXCR4/Alexa 647-conjugated anti-rabbit and anti-ACKR3/Alexa 488-conjugated anti-mouse and analyzed for receptor expression via flow cytometry. RFU: relative fluorescence units. The horizontal and vertical lines show the gating thresholds for CXCR4 (Alexa 647) and ACKR3 (Alexa 488). B-E . Quantification of CXCR4 ( B/C ) and ACKR3 ( D/E ) cell surface expression by flow cytometry. Cells were stimulated with 100 μM cyclazosin ( B/D ) or prazosin ( C/E ) as indicated. Data are mean ± SE from 4 independent experiments. *: p

    Journal: PLoS ONE

    Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

    doi: 10.1371/journal.pone.0204041

    Figure Lengend Snippet: Prazosin and cyclazosin induce internalization of CXCR4 and ACKR3 in hVSMCs. A. hVSMC were treated with 100 μM cyclazosin or prazosin at 37°C for 15 or 30 min, stained with anti-CXCR4/Alexa 647-conjugated anti-rabbit and anti-ACKR3/Alexa 488-conjugated anti-mouse and analyzed for receptor expression via flow cytometry. RFU: relative fluorescence units. The horizontal and vertical lines show the gating thresholds for CXCR4 (Alexa 647) and ACKR3 (Alexa 488). B-E . Quantification of CXCR4 ( B/C ) and ACKR3 ( D/E ) cell surface expression by flow cytometry. Cells were stimulated with 100 μM cyclazosin ( B/D ) or prazosin ( C/E ) as indicated. Data are mean ± SE from 4 independent experiments. *: p

    Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

    Techniques: Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence

    Prazosin and cyclazosin induce chemical shift changes in the NMR spectra of CXCR4 and ACKR3 in membranes. 1 H- 13 C HSQC spectra of reductively methylated CXCR4 ( A-C ) and ACKR3 ( D-F ) membrane preparations were recorded without (black) and with (red) 200 μM prazosin ( A/D ), cyclazosin ( B/E ) or atipamezol ( C/F ). Black arrows indicate significant differences in chemical shifts or broadening (loss) of the signal.

    Journal: PLoS ONE

    Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

    doi: 10.1371/journal.pone.0204041

    Figure Lengend Snippet: Prazosin and cyclazosin induce chemical shift changes in the NMR spectra of CXCR4 and ACKR3 in membranes. 1 H- 13 C HSQC spectra of reductively methylated CXCR4 ( A-C ) and ACKR3 ( D-F ) membrane preparations were recorded without (black) and with (red) 200 μM prazosin ( A/D ), cyclazosin ( B/E ) or atipamezol ( C/F ). Black arrows indicate significant differences in chemical shifts or broadening (loss) of the signal.

    Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

    Techniques: Nuclear Magnetic Resonance, Methylation

    CXCL12-inudced ERK1/2 phosphorylation in HEK293 cells is augmented after transfection with CXCR4 and ACKR3. HEK293 cells were transfected with empty vector (left), CXCR4 (center) or ACKR3 (right) and stimulated with 100 nM of CXCL12 for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2. The migration position of molecular mass standards is indicated.

    Journal: PLoS ONE

    Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

    doi: 10.1371/journal.pone.0204041

    Figure Lengend Snippet: CXCL12-inudced ERK1/2 phosphorylation in HEK293 cells is augmented after transfection with CXCR4 and ACKR3. HEK293 cells were transfected with empty vector (left), CXCR4 (center) or ACKR3 (right) and stimulated with 100 nM of CXCL12 for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2. The migration position of molecular mass standards is indicated.

    Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Migration

    Dose-dependent effects of prazosin and cyclazosin in PRESTO-Tango β-arrestin recruitment assays for CXCR4 and ACKR3. Data are mean ± SE from n = 3 independent experiments (in triplicates). RLU: relative luminescence units. A-C: CXCR4 PRESTO-Tango assays. A. Grey circles: cells treated with cyclazosin. Black circles: cells treated with prazosin. B. Cells were pre-incubated with vehicle (control, grey circles) or AMD3100 (10 μM; black circles) for 15 min, followed by stimulation with cyclazosin. **: p

    Journal: PLoS ONE

    Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

    doi: 10.1371/journal.pone.0204041

    Figure Lengend Snippet: Dose-dependent effects of prazosin and cyclazosin in PRESTO-Tango β-arrestin recruitment assays for CXCR4 and ACKR3. Data are mean ± SE from n = 3 independent experiments (in triplicates). RLU: relative luminescence units. A-C: CXCR4 PRESTO-Tango assays. A. Grey circles: cells treated with cyclazosin. Black circles: cells treated with prazosin. B. Cells were pre-incubated with vehicle (control, grey circles) or AMD3100 (10 μM; black circles) for 15 min, followed by stimulation with cyclazosin. **: p

    Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

    Techniques: Incubation

    Re-organization of α 1A/B/D -AR heteromers after depletion of CXCR4 or ACKR3 in hVSMCs. Quantification of PLA signals for the detection of receptor-receptor interactions in hVSMCs after incubation with non-targeting (NT) siRNA, CXCR4 siRNA, or ACKR3 siRNA, as in Fig 5 . N = 3 independent experiments with n = 10 images per experiment. *: p

    Journal: PLoS ONE

    Article Title: Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

    doi: 10.1371/journal.pone.0253821

    Figure Lengend Snippet: Re-organization of α 1A/B/D -AR heteromers after depletion of CXCR4 or ACKR3 in hVSMCs. Quantification of PLA signals for the detection of receptor-receptor interactions in hVSMCs after incubation with non-targeting (NT) siRNA, CXCR4 siRNA, or ACKR3 siRNA, as in Fig 5 . N = 3 independent experiments with n = 10 images per experiment. *: p

    Article Snippet: To visualize proteins individually, slides were incubated with rabbit anti-AVPR1A (Bioss BS-11598R), mouse anti-ACKR3 (R & D MAB42273), goat anti-CXCR4 (Abcam Ab1670) or rabbit anti-CXCR4 (Alomone Labs ACR-014), mouse anti-α1A -AR (Abcam Ab87990) or rabbit anti-α1A -AR (Abcam Ab137123), rabbit anti-α1B -AR (Abcam Ab169523) or goat anti-α1B -AR (Santa Cruz SC27136), and goat anti-α1D-AR (Santa Cruz SC27099) at 37°C for 105 min in a humidifying chamber.

    Techniques: Proximity Ligation Assay, Incubation

    Re-organization of heteromers between α 1A/B/D -ARs, CXCR4, and ACKR3 upon depletion of AVPR1A in hVSMCs. Quantification of PLA signals for the detection of receptor-receptor interactions in hVSMCs after incubation with non-targeting (NT) siRNA or AVPR1A siRNA, as in Fig 8 . N = 4 independent experiments with n = 10 images per experiment. ΔPLA signals (%NT): Change in PLA signals in cells incubated with AVPR1A siRNA in % of PLA signals in cells incubated with NT siRNA (= 100%). *: p

    Journal: PLoS ONE

    Article Title: Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

    doi: 10.1371/journal.pone.0253821

    Figure Lengend Snippet: Re-organization of heteromers between α 1A/B/D -ARs, CXCR4, and ACKR3 upon depletion of AVPR1A in hVSMCs. Quantification of PLA signals for the detection of receptor-receptor interactions in hVSMCs after incubation with non-targeting (NT) siRNA or AVPR1A siRNA, as in Fig 8 . N = 4 independent experiments with n = 10 images per experiment. ΔPLA signals (%NT): Change in PLA signals in cells incubated with AVPR1A siRNA in % of PLA signals in cells incubated with NT siRNA (= 100%). *: p

    Article Snippet: To visualize proteins individually, slides were incubated with rabbit anti-AVPR1A (Bioss BS-11598R), mouse anti-ACKR3 (R & D MAB42273), goat anti-CXCR4 (Abcam Ab1670) or rabbit anti-CXCR4 (Alomone Labs ACR-014), mouse anti-α1A -AR (Abcam Ab87990) or rabbit anti-α1A -AR (Abcam Ab137123), rabbit anti-α1B -AR (Abcam Ab169523) or goat anti-α1B -AR (Santa Cruz SC27136), and goat anti-α1D-AR (Santa Cruz SC27099) at 37°C for 105 min in a humidifying chamber.

    Techniques: Proximity Ligation Assay, Incubation

    siRNA knockdown of CXCR4 and ACKR3 in hVSMCs. ( A ) Representative PLA images for the detection of CXCR4 in hVSMC after incubation with non-targeting (NT) or CXCR4 siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. ( B ) Representative PLA images for the detection of ACKR3 in hVSMC after incubation with NT or ACKR3 siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm. ( C ) Quantification of PLA signals for the detection of CXCR4 and ACKR3 after incubation with siRNA, as in A/B. N = 3 independent experiments with n = 10 images per experiment. *: p

    Journal: PLoS ONE

    Article Title: Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

    doi: 10.1371/journal.pone.0253821

    Figure Lengend Snippet: siRNA knockdown of CXCR4 and ACKR3 in hVSMCs. ( A ) Representative PLA images for the detection of CXCR4 in hVSMC after incubation with non-targeting (NT) or CXCR4 siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. ( B ) Representative PLA images for the detection of ACKR3 in hVSMC after incubation with NT or ACKR3 siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm. ( C ) Quantification of PLA signals for the detection of CXCR4 and ACKR3 after incubation with siRNA, as in A/B. N = 3 independent experiments with n = 10 images per experiment. *: p

    Article Snippet: To visualize proteins individually, slides were incubated with rabbit anti-AVPR1A (Bioss BS-11598R), mouse anti-ACKR3 (R & D MAB42273), goat anti-CXCR4 (Abcam Ab1670) or rabbit anti-CXCR4 (Alomone Labs ACR-014), mouse anti-α1A -AR (Abcam Ab87990) or rabbit anti-α1A -AR (Abcam Ab137123), rabbit anti-α1B -AR (Abcam Ab169523) or goat anti-α1B -AR (Santa Cruz SC27136), and goat anti-α1D-AR (Santa Cruz SC27099) at 37°C for 105 min in a humidifying chamber.

    Techniques: Proximity Ligation Assay, Incubation

    Detection of heteromers between α 1A/B/D -ARs, CXCR4, and ACKR3 upon depletion of AVPR1A in hVSMCs. Representative PLA images for the detection of receptor-receptor interactions in hVSMC after incubation with non-targeting (NT) siRNA or AVPR1A siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm.

    Journal: PLoS ONE

    Article Title: Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

    doi: 10.1371/journal.pone.0253821

    Figure Lengend Snippet: Detection of heteromers between α 1A/B/D -ARs, CXCR4, and ACKR3 upon depletion of AVPR1A in hVSMCs. Representative PLA images for the detection of receptor-receptor interactions in hVSMC after incubation with non-targeting (NT) siRNA or AVPR1A siRNA. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm.

    Article Snippet: To visualize proteins individually, slides were incubated with rabbit anti-AVPR1A (Bioss BS-11598R), mouse anti-ACKR3 (R & D MAB42273), goat anti-CXCR4 (Abcam Ab1670) or rabbit anti-CXCR4 (Alomone Labs ACR-014), mouse anti-α1A -AR (Abcam Ab87990) or rabbit anti-α1A -AR (Abcam Ab137123), rabbit anti-α1B -AR (Abcam Ab169523) or goat anti-α1B -AR (Santa Cruz SC27136), and goat anti-α1D-AR (Santa Cruz SC27099) at 37°C for 105 min in a humidifying chamber.

    Techniques: Proximity Ligation Assay, Incubation

    Detection of α 1A/B/D -AR heteromers after depletion of CXCR4 or ACKR3 in hVSMCs. Representative PLA images for the detection of α 1A/B/D -AR heteromers in hVSMCs after incubation with non-targeting (NT) siRNA (left), CXCR4 siRNA (center), or ACKR3 siRNA (right). Ctrl: Omission of one primary antibody. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm.

    Journal: PLoS ONE

    Article Title: Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

    doi: 10.1371/journal.pone.0253821

    Figure Lengend Snippet: Detection of α 1A/B/D -AR heteromers after depletion of CXCR4 or ACKR3 in hVSMCs. Representative PLA images for the detection of α 1A/B/D -AR heteromers in hVSMCs after incubation with non-targeting (NT) siRNA (left), CXCR4 siRNA (center), or ACKR3 siRNA (right). Ctrl: Omission of one primary antibody. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Scale bar = 10 μm.

    Article Snippet: To visualize proteins individually, slides were incubated with rabbit anti-AVPR1A (Bioss BS-11598R), mouse anti-ACKR3 (R & D MAB42273), goat anti-CXCR4 (Abcam Ab1670) or rabbit anti-CXCR4 (Alomone Labs ACR-014), mouse anti-α1A -AR (Abcam Ab87990) or rabbit anti-α1A -AR (Abcam Ab137123), rabbit anti-α1B -AR (Abcam Ab169523) or goat anti-α1B -AR (Santa Cruz SC27136), and goat anti-α1D-AR (Santa Cruz SC27099) at 37°C for 105 min in a humidifying chamber.

    Techniques: Proximity Ligation Assay, Incubation

    Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

    doi: 10.1161/JAHA.117.006575

    Figure Lengend Snippet: Peptides derived from transmembrane domains of atypical chemokine receptor 3 (ACKR3) alter receptor heteromerization in human vascular smooth muscle cells (hVSMCs). hVSMCs were treated with vehicle or transmembrane (TM) 2/4/7 peptide analogs (10 μmol/L, 30 minutes at 37°C), washed and used for proximity ligation assay (PLA). A, Typical PLA images for the detection of individual receptors. Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of receptor‐receptor interactions. Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. AR indicates adrenergic receptor; CXCR4, chemokine (C‐X‐C motif) receptor 4.

    Article Snippet: To visualize receptor‐receptor interactions, slides were incubated with a combination of rabbit anti‐HA (Abcam 9110) and mouse anti‐FLAG (Sigma F1804), mouse anti‐ACKR3 (R & D MAB42273), and goat anti‐CXCR4 (Abcam 1670) or rabbit anti‐α1A ‐AR (Abcam Ab137123) or rabbit anti‐α1B ‐AR (Abcam Ab169523) or goat anti‐α1D ‐AR (Santa Cruz SC27099) or with a combination of goat anti‐CXCR4 (Abcam 1670) and rabbit anti‐α1A ‐AR (Abcam Ab137123) or rabbit anti‐α1B ‐AR (Abcam Ab169523) or rabbit anti‐CXCR4 (Alomone Labs ACR‐014) and goat anti‐ α1D ‐AR (Santa Cruz SC27099) at 37°C for 105 minutes in a humidifying chamber.

    Techniques: Derivative Assay, Proximity Ligation Assay

    Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

    doi: 10.1161/JAHA.117.006575

    Figure Lengend Snippet: Hetero‐oligomerization of α 1 ‐adrenergic receptor (AR) with atypical chemokine receptor 3 (ACKR3) and chemokine (C‐X‐C motif) receptor 4 (CXCR4)—working model. A, Hetero‐oligomeric complexes between α 1B/D ‐ AR and the ACKR 3: CXCR 4 heteromer are responsible for α 1B/D ‐ AR signaling on catecholamine stimulation in human vascular smooth muscle cells (hVSMCs). Activation of ACKR 3 inhibits, whereas activation of CXCR 4 sensitizes this response via allosteric modulation of α 1B/D ‐ AR within the hetero‐oligomeric receptor complex. Disruption of the α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomeric complex inactivates α 1B/D ‐ AR signaling and shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium, which leads to the assembly of newly formed heteromeric complexes between α 1A/B ‐ AR and the ACKR 3 and/or CXCR 4 protomers/homodimers. B, Disruption of ACKR 3: CXCR 4 heteromers with peptide analogs of transmembrane ( TM ) domains of ACKR 3 leads to the assembly of newly formed heteromeric receptor complexes. The TM 2 peptide analog interferes with ACKR 3: CXCR 4 and α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, which inhibits α 1B/D ‐ AR signaling/function and leads to the formation of increased numbers of α 1A/B ‐ AR : CXCR 4 complexes, and presumably to increased numbers of ACKR 3 protomers/homodimers (not shown). The TM 7 peptide disrupts ACKR 3: CXCR 4 heteromers without affecting α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomerization, leading to increases in α 1A ‐ AR : ACKR 3 and α 1A ‐ AR : CXCR 4 heteromers. Because α 1B/D ‐ AR : ACKR 3: CXCR 4 hetero‐oligomers are not affected by the TM 7 peptide, α 1B/D ‐ AR signaling/function is maintained.

    Article Snippet: To visualize receptor‐receptor interactions, slides were incubated with a combination of rabbit anti‐HA (Abcam 9110) and mouse anti‐FLAG (Sigma F1804), mouse anti‐ACKR3 (R & D MAB42273), and goat anti‐CXCR4 (Abcam 1670) or rabbit anti‐α1A ‐AR (Abcam Ab137123) or rabbit anti‐α1B ‐AR (Abcam Ab169523) or goat anti‐α1D ‐AR (Santa Cruz SC27099) or with a combination of goat anti‐CXCR4 (Abcam 1670) and rabbit anti‐α1A ‐AR (Abcam Ab137123) or rabbit anti‐α1B ‐AR (Abcam Ab169523) or rabbit anti‐CXCR4 (Alomone Labs ACR‐014) and goat anti‐ α1D ‐AR (Santa Cruz SC27099) at 37°C for 105 minutes in a humidifying chamber.

    Techniques: Activation Assay

    α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

    doi: 10.1161/JAHA.117.006575

    Figure Lengend Snippet: α 1B/D ‐Adrenergic receptors (ARs) form hetero‐oligomeric complexes with the atypical chemokine receptor 3 (ACKR3): chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromer in human vascular smooth muscle cells (hVSMCs). A, Typical proximity ligation assay (PLA) images for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride ( DAPI ) signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Typical PLA images for the detection of CXCR 4 and ACKR 3:α 1A/B/D ‐ AR heteromers in hVSMCs incubated with NT or CXCR 4 si RNA . Images show merged PLA / DAPI signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. C, Quantification of PLA signals per cell for the detection of ACKR 3 and CXCR 4:α 1A/B/D ‐ AR heteromers, as in (A). n=3 independent experiments with n=10 images per condition and experiment. * P

    Article Snippet: To visualize receptor‐receptor interactions, slides were incubated with a combination of rabbit anti‐HA (Abcam 9110) and mouse anti‐FLAG (Sigma F1804), mouse anti‐ACKR3 (R & D MAB42273), and goat anti‐CXCR4 (Abcam 1670) or rabbit anti‐α1A ‐AR (Abcam Ab137123) or rabbit anti‐α1B ‐AR (Abcam Ab169523) or goat anti‐α1D ‐AR (Santa Cruz SC27099) or with a combination of goat anti‐CXCR4 (Abcam 1670) and rabbit anti‐α1A ‐AR (Abcam Ab137123) or rabbit anti‐α1B ‐AR (Abcam Ab169523) or rabbit anti‐CXCR4 (Alomone Labs ACR‐014) and goat anti‐ α1D ‐AR (Santa Cruz SC27099) at 37°C for 105 minutes in a humidifying chamber.

    Techniques: Proximity Ligation Assay, Incubation, Small Interfering RNA

    Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

    doi: 10.1161/JAHA.117.006575

    Figure Lengend Snippet: Atypical chemokine receptor 3 (ACKR3) gene silencing reduces ACKR 3:α 1B/D ‐adrenergic receptor (AR) and ACKR 3: chemokine (C‐X‐C motif) receptor 4 (CXCR4) heteromerization and inhibits α 1B/D ‐ AR signaling in human vascular smooth muscle cells (hVSMCs). A, Representative proximity ligation assay (PLA) images for the detection of individual receptors (left) and receptor‐receptor interactions (right) in hVSMCs incubated with nontargeting ( NT ) or ACKR 3 small interfering RNA (siRNA). Images show merged PLA /4′,6‐diamidino‐2‐phenylindole dihydrochloride signals. Ctrl: Omission of one primary antibody. Scale bars=10 μm. B, Quantification of PLA signals per cell for the detection of individual receptors, as in (A). n=4 independent experiments with n=10 images per condition and experiment. * P

    Article Snippet: To visualize receptor‐receptor interactions, slides were incubated with a combination of rabbit anti‐HA (Abcam 9110) and mouse anti‐FLAG (Sigma F1804), mouse anti‐ACKR3 (R & D MAB42273), and goat anti‐CXCR4 (Abcam 1670) or rabbit anti‐α1A ‐AR (Abcam Ab137123) or rabbit anti‐α1B ‐AR (Abcam Ab169523) or goat anti‐α1D ‐AR (Santa Cruz SC27099) or with a combination of goat anti‐CXCR4 (Abcam 1670) and rabbit anti‐α1A ‐AR (Abcam Ab137123) or rabbit anti‐α1B ‐AR (Abcam Ab169523) or rabbit anti‐CXCR4 (Alomone Labs ACR‐014) and goat anti‐ α1D ‐AR (Santa Cruz SC27099) at 37°C for 105 minutes in a humidifying chamber.

    Techniques: Proximity Ligation Assay, Incubation, Small Interfering RNA