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Concentration-response relationships of ( A ) DAMGO as a recruiter of β-arrestin2 to human μ receptors in PathHunter CHO cells and ( B ) DAMGO as an inhibitor of forskolin (30 μM) stimulated cAMP accumulation in the same cell line transiently expressing the pGloSensor-22F protein following 16 h exposure to 1 μM PP2, <t>PP3,</t> eCF506 or an equal volume of DMSO. (A) Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each 96-well plate. The insert demonstrates c-Src phosphorylation at Y416 (upper panel) and total c-Src (lower panel) expression assessed using western blot in each cell lysate following overnight exposure and confirms inhibition of c-Src phosphorylation. (B) Data are expressed as a percentage of luminescence in each well measured prior to the addition of agonist (% control). Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 /IC 50 ) parameters, presented in . Data are the mean ± SEM of 5 replicates (A) and 8 replicates (B).
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Concentration-response relationships of ( A ) DAMGO as a recruiter of β-arrestin2 to human μ receptors in PathHunter CHO cells and ( B ) DAMGO as an inhibitor of forskolin (30 μM) stimulated cAMP accumulation in the same cell line transiently expressing the pGloSensor-22F protein following 16 h exposure to 1 μM PP2, <t>PP3,</t> eCF506 or an equal volume of DMSO. (A) Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each 96-well plate. The insert demonstrates c-Src phosphorylation at Y416 (upper panel) and total c-Src (lower panel) expression assessed using western blot in each cell lysate following overnight exposure and confirms inhibition of c-Src phosphorylation. (B) Data are expressed as a percentage of luminescence in each well measured prior to the addition of agonist (% control). Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 /IC 50 ) parameters, presented in . Data are the mean ± SEM of 5 replicates (A) and 8 replicates (B).
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Concentration-response relationships of ( A ) DAMGO as a recruiter of β-arrestin2 to human μ receptors in PathHunter CHO cells and ( B ) DAMGO as an inhibitor of forskolin (30 μM) stimulated cAMP accumulation in the same cell line transiently expressing the pGloSensor-22F protein following 16 h exposure to 1 μM PP2, <t>PP3,</t> eCF506 or an equal volume of DMSO. (A) Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each 96-well plate. The insert demonstrates c-Src phosphorylation at Y416 (upper panel) and total c-Src (lower panel) expression assessed using western blot in each cell lysate following overnight exposure and confirms inhibition of c-Src phosphorylation. (B) Data are expressed as a percentage of luminescence in each well measured prior to the addition of agonist (% control). Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 /IC 50 ) parameters, presented in . Data are the mean ± SEM of 5 replicates (A) and 8 replicates (B).
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Concentration-response relationships of ( A ) DAMGO as a recruiter of β-arrestin2 to human μ receptors in PathHunter CHO cells and ( B ) DAMGO as an inhibitor of forskolin (30 μM) stimulated cAMP accumulation in the same cell line transiently expressing the pGloSensor-22F protein following 16 h exposure to 1 μM PP2, PP3, eCF506 or an equal volume of DMSO. (A) Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each 96-well plate. The insert demonstrates c-Src phosphorylation at Y416 (upper panel) and total c-Src (lower panel) expression assessed using western blot in each cell lysate following overnight exposure and confirms inhibition of c-Src phosphorylation. (B) Data are expressed as a percentage of luminescence in each well measured prior to the addition of agonist (% control). Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 /IC 50 ) parameters, presented in . Data are the mean ± SEM of 5 replicates (A) and 8 replicates (B).

Journal: bioRxiv

Article Title: The c-Src inhibitor eCF506 diminishes opioid tolerance creating bias against β-arrestin2 recruitment

doi: 10.1101/2025.07.01.662507

Figure Lengend Snippet: Concentration-response relationships of ( A ) DAMGO as a recruiter of β-arrestin2 to human μ receptors in PathHunter CHO cells and ( B ) DAMGO as an inhibitor of forskolin (30 μM) stimulated cAMP accumulation in the same cell line transiently expressing the pGloSensor-22F protein following 16 h exposure to 1 μM PP2, PP3, eCF506 or an equal volume of DMSO. (A) Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each 96-well plate. The insert demonstrates c-Src phosphorylation at Y416 (upper panel) and total c-Src (lower panel) expression assessed using western blot in each cell lysate following overnight exposure and confirms inhibition of c-Src phosphorylation. (B) Data are expressed as a percentage of luminescence in each well measured prior to the addition of agonist (% control). Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 /IC 50 ) parameters, presented in . Data are the mean ± SEM of 5 replicates (A) and 8 replicates (B).

Article Snippet: DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) and forskolin (HelloBio, UK), morphine sulfate salt pentahydrate (#M8777), fentanyl citrate (#F3886), oxycodone hydrochloride (#BP1068; all from Merck, UK), TRV130 (#MBS3601671; CliniSciences, UK), buprenorphine hydrochloride (#2808), β-FNA (#0926), PP2 (#1407), PP3 (#2794), caffeic acid pYEEIE (#1935; all from Tocris, UK), DAS-5-oCRBN (#HY-163144; Cambridge Biosciences, UK) were prepared as 10 mM (DAMGO, morphine, fentanyl, buprenorphine, TRV130, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506), 20 mM (oxycodone), 25 mM (forskolin) or 100 mM (β-FNA) stock solutions in DMSO (DAMGO, forskolin, buprenorphine, β-FNA, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506) or water (fentanyl, morphine, oxycodone).

Techniques: Concentration Assay, Expressing, Produced, Phospho-proteomics, Western Blot, Inhibition, Control

Journal: bioRxiv

Article Title: The c-Src inhibitor eCF506 diminishes opioid tolerance creating bias against β-arrestin2 recruitment

doi: 10.1101/2025.07.01.662507

Figure Lengend Snippet:

Article Snippet: DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) and forskolin (HelloBio, UK), morphine sulfate salt pentahydrate (#M8777), fentanyl citrate (#F3886), oxycodone hydrochloride (#BP1068; all from Merck, UK), TRV130 (#MBS3601671; CliniSciences, UK), buprenorphine hydrochloride (#2808), β-FNA (#0926), PP2 (#1407), PP3 (#2794), caffeic acid pYEEIE (#1935; all from Tocris, UK), DAS-5-oCRBN (#HY-163144; Cambridge Biosciences, UK) were prepared as 10 mM (DAMGO, morphine, fentanyl, buprenorphine, TRV130, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506), 20 mM (oxycodone), 25 mM (forskolin) or 100 mM (β-FNA) stock solutions in DMSO (DAMGO, forskolin, buprenorphine, β-FNA, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506) or water (fentanyl, morphine, oxycodone).

Techniques: Inhibition, Concentration Assay, cAMP Assay

Concentration-response relationships of DAMGO to recruit β-arrestin2 to human μ receptors in PathHunter CHO cells transiently overexpressing ( A ) full-length (residues 1-536; WT), ( B ) C-terminally truncated (residues 1-249) or ( C ) N-terminally truncated and catalytically inactive (residues 250-536(K298M)) human c-Src following 16-h exposure to PP2, PP3, eCF506 (300 nM) or an equal volume of DMSO. Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each plate i.e., cells overexpressing (A) WT, (B) Src 1-249 or (C) Src 250-536(K298M) are each expressed relative to the maximum β-arrestin2 recruitment of each construct caused by DAMGO. Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 ) parameters, presented in Supplementary Table 3. Data are the mean ± SEM of 5-6 replicates.

Journal: bioRxiv

Article Title: The c-Src inhibitor eCF506 diminishes opioid tolerance creating bias against β-arrestin2 recruitment

doi: 10.1101/2025.07.01.662507

Figure Lengend Snippet: Concentration-response relationships of DAMGO to recruit β-arrestin2 to human μ receptors in PathHunter CHO cells transiently overexpressing ( A ) full-length (residues 1-536; WT), ( B ) C-terminally truncated (residues 1-249) or ( C ) N-terminally truncated and catalytically inactive (residues 250-536(K298M)) human c-Src following 16-h exposure to PP2, PP3, eCF506 (300 nM) or an equal volume of DMSO. Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each plate i.e., cells overexpressing (A) WT, (B) Src 1-249 or (C) Src 250-536(K298M) are each expressed relative to the maximum β-arrestin2 recruitment of each construct caused by DAMGO. Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 ) parameters, presented in Supplementary Table 3. Data are the mean ± SEM of 5-6 replicates.

Article Snippet: DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) and forskolin (HelloBio, UK), morphine sulfate salt pentahydrate (#M8777), fentanyl citrate (#F3886), oxycodone hydrochloride (#BP1068; all from Merck, UK), TRV130 (#MBS3601671; CliniSciences, UK), buprenorphine hydrochloride (#2808), β-FNA (#0926), PP2 (#1407), PP3 (#2794), caffeic acid pYEEIE (#1935; all from Tocris, UK), DAS-5-oCRBN (#HY-163144; Cambridge Biosciences, UK) were prepared as 10 mM (DAMGO, morphine, fentanyl, buprenorphine, TRV130, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506), 20 mM (oxycodone), 25 mM (forskolin) or 100 mM (β-FNA) stock solutions in DMSO (DAMGO, forskolin, buprenorphine, β-FNA, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506) or water (fentanyl, morphine, oxycodone).

Techniques: Concentration Assay, Produced, Construct