1. GRK2 Constitutively Governs Peripheral Delta Opioid Receptor Activity
Allison Doyle Brackley, Ruben Gomez, Armen N. Akopian, Michael A. Henry, Nathaniel A. Jeske 
Cell Reports 
Volume 16, Issue 10, Pages (September 2016)
DOI: /j.celrep
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2. Cell Reports 2016 16, 2686-2698DOI: (10.1016/j.celrep.2016.07.084)
Copyright © 2016 The Author(s) Terms and Conditions

3. Figure 1 Functional DOR Competence in Sensory Neurons
(A and B) Dose response for DPDPE inhibition of KCl (50 mM)-evoked Ca2+ influx in (A) TG or (B) DRG pretreated with vehicle or BK (200 nM, 5 min) following 2 hr serum-starvation. ∗∗∗p < versus vehicle; (A) n = 26–57 and (B) n = 17–39 neurons/dose; two-way ANOVA Bonferroni post hoc; mean ± SEM. Least-squares fit (best-fit) variable slope curves used to determine IC50 for vehicle (dotted line) and BK (solid line) and 95% confidence intervals (gray).
(C) Effect of naltrindole pretreatment on DPDPE (1 μM) inhibition of KCl-evoked Ca2+ influx in DRG neurons pretreated vehicle or naltrindole (10 nM, 5 min) prior to vehicle or BK (200 nM, 5 min) treatment following 2 hr serum-starvation. ∗∗p < 0.01; ns, no significance; n = 14–28 DRG/group; two-way ANOVA Bonferroni post hoc; mean ± SEM.
(D) Comparison of DPDPE (1 μM) versus SNC 80 (1 μM) inhibition of KCl-evoked Ca2+ influx in DRG neurons pretreated with vehicle or BK (200 nM, 5 min) following 2 hr serum-starvation. ∗∗∗p < 0.005; n = 24–34 DRG/group; two-way ANOVA Bonferroni post hoc; mean ± SEM.
(E and F) Representative traces (E) and quantification (F) of DPDPE (1 μM) inhibition of VGCCs in DRG neurons (20–35 pF) pretreated with vehicle or BK (200 nM, 10 min) following 2 hr serum-starvation. ∗∗∗p < 0.005; n = 4–9 DRG/group; one-way ANOVA Bonferroni post hoc; mean ± SEM.
See also Figures S1 and S2.
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4. Figure 2 GRK2 Modulation of DOR Activity
(A) Crude PM coIP from TG cultures serum-starved for 18 hr and treated with vehicle or BK (200 nM, 5 min). ∗p < 0.05; n = 3 independent trials; unpaired two-tailed Student’s t test; mean ± SEM.
(B) WCL from 2 hr serum-starved TG cultures transfected in mock fashion or with mismatch, GRK2, or FITC-GRK2 siRNA. ∗∗∗p < 0.005; ns, no significance; n = 6 independent trials; one-way ANOVA Bonferroni post hoc; mean ± SEM.
(C and D) Cumulative traces (C) and quantification (D) of DPDPE (1 μM) inhibition of KCl (50 mM)-evoked Ca2+ influx in DRG (mock-treated or transfected with FITC-GRK2 siRNA) pre-treated with vehicle or BK (200 nM, 5 min) following 2 hr serum-starvation. ∗∗∗p < 0.005; n = 22–37 DRG/group; two-way ANOVA Bonferroni post hoc; mean ± SEM.
(E) DPDPE (1 μM) inhibition of VGCCs in DRG (mock-treated or transfected with FITC-GRK2 siRNA) pre-treated with vehicle or BK (200 nM, 10 min) following 2 hr serum-starvation. ∗∗∗p < 0.005; n = 5–8 DRG/group; two-way ANOVA Bonferroni post hoc; mean ± SEM.
See also Figure S3.
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5. Figure 3
Constitutive DOR Incompetence Is Independent of GRK2 Kinase Activity
(A) DOR phosphorylation at Ser363 in crude PM immunoprecipitates from TG cultures serum-starved for 18 hr and treated with vehicle or BK (200 nM, 5 min). ns, no significance; n = 3 independent trials; unpaired two-tailed Student’s t test; mean ± SEM.
(B and C) Cumulative traces (B) and quantification (C) of DPDPE (1 μM) inhibition of KCl (50 mM)-evoked Ca2+ influx in nucleofected DRG (overexpression: empty vector [E.V.] + GFP, GRK2 + GFP, K220R + GFP cDNAs) pretreated with vehicle or BK (200 nM, 5 min) following 2 hr serum-starvation. ∗p < 0.05; n = 20–31 DRG/group; two-way ANOVA Bonferroni post hoc; mean ± SEM.
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6. Figure 4 GRK2 Modulation of DOR-Mediated Antinociception
(A) Timeline for ODN injections and rat behavior protocol for BK priming of peripheral DOR antinociception.
(B) WCL from ipsilateral (ipsi-) and contralateral (contra-) DRG of rats treated with MM-ODN or GRK2 AS-ODN. ∗p < 0.05; ns, no significance; n = 3 independent trials; one-way ANOVA Bonferroni post hoc; mean ± SEM.
(C–F) Time course for DPDPE inhibition of PGE2-induced allodynia in ipsilateral (C), mechanical; (D), thermal and contralateral (E), mechanical; (F), thermal hindpaws. Paw withdrawal readings were measured 5 min and 10 min post-intraplantar (i.pl.) injection (vehicle or BK [25 μg], and 5-min intervals for 20 min following second i.pl. injection (co-injection DPDPE [20 μg]/PGE2 [0.3 μg]); BK-induced allodynia: mechanical and thermal, ∗∗∗p < versus vehicle-treated groups; DPDPE inhibition of PGE2-induced allodynia: mechanical, 20 min, ∗p < 0.05 (MM-ODN/BK), 25 min, ∗∗p < 0.01 (AS-ODN/BK), ∗∗∗p < (AS-ODN/Veh, MM-ODN/BK); 30 min, ∗p < 0.05 (AS-ODN/Veh), ∗∗p < 0.01 (AS-ODN/BK, MM-ODN/BK); 35 min, ∗∗∗p < (MM-ODN/BK, AS-ODN/Veh, AS-ODN/BK) versus MM-ODN/Veh; thermal, ∗∗p < 0.01 MM-ODN/BK versus AS-ODN/Veh; 25–35, ∗∗∗p < versus groups below baseline (BL) readings; n = 6 rats per group; two-way ANOVA Bonferroni post hoc; mean ± SEM.
(G and H) Quantified antinociceptive effect of DPDPE at 35 min for mechanical (G) and thermal (H) readings. ∗∗∗p < 0.005; ns, no significance; n = 6 rats per group; two-way ANOVA Bonferroni post hoc; mean ± SEM.
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7. Figure 5 BK Modulation of RKIP Signaling in Sensory Neurons
(A) Immunohistochemical expression of RKIP (red) in rat TG and DRG with Topro (blue) to identify nuclei. Scale bars, 50 μm (yellow) and 15 μm (white). Confocal images are representative of four independent trials.
(B) Cytosolic lysates from TG cultures serum-starved for 18 hr and treated with vehicle (5 min)/vehicle (5 min), BK (200 nM, 5 min)/vehicle, BK/HOE-140 (10 μM, 5 min), or BK/GFX (GF X, 10 μM, 5 min). ∗∗p < 0.01 versus vehicle/vehicle, ns, no significance; n = 3 independent trials; one-way ANOVA Bonferroni post hoc; mean ± SEM.
(C) Cytosolic coIP from TG cultures serum-starved for 18 hr and treated with vehicle (5 min)/vehicle (5 min), vehicle/BK (200 nM, 5 min), or BK/GFX (GF X, 10 μM, 5 min). ∗p < 0.05; ns = no significance; n = 3 independent trials; one-way ANOVA Bonferroni post hoc; mean ± SEM.
See also Figure S4.
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8. Figure 6 RKIP Modulation of Functional DOR Competence
(A) WCL from 2 hr serum-starved TG cultures transfected in mock fashion or with mismatch, RKIP, or FITC-RKIP siRNA. ∗p < 0.05, ∗∗p < 0.01, ns, no significance versus mock; n = 3 independent trials; one-way ANOVA Bonferroni post hoc; mean ± SEM.
(B and C) Cumulative traces (B) and quantification (C) of DPDPE (1 μM) inhibition of KCl (50 mM)-evoked Ca2+ influx in DRG (mock-treated, transfected with FITC-RKIP siRNA or FITC-RKIP siRNA followed by nucleofection RKIP and GFP cDNAs [RKIP Rescue]) pretreated with vehicle or BK (200 nM, 5 min) following 2 hr serum-starvation. ∗∗p < 0.01; ns, no significance; n = 20–26 DRG/group; two-way ANOVA Bonferroni post hoc; mean ± SEM.
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9. Figure 7 GRK2 Hinders Peripheral DOR G Protein Coupling
(A and B) Crude PM coIP from TG cultures serum-starved for 18 hr and treated with vehicle or BK (200 nM, 5 min) (A) or vehicle or BK (200 nM, 5 min) then DPDPE (1 μM, 15 min) (B). (A) ∗p < 0.05; ns, no significance; n = 3 independent trials; unpaired two-tailed Student’s t test; mean ± SEM.
(C) Crude PM coIP from TG cultures serum-starved for 2 hr following mock- or GRK2-siRNA nucleofection and treated with vehicle or BK (200 nM, 5 min). ∗p < 0.05; n = 3 independent trials; two-way ANOVA Bonferroni post hoc; mean ± SEM.
(D) Crude PM coIP from TG cultures co-treated for 18 hr with empty vector (E.V.) or GRK2 cDNA nucleofection (overexpression) in serum-starved media followed by treatment with vehicle or BK (200 nM, 5 min). ∗p < 0.05; ns, no significance; n = 3 independent trials; unpaired two-tailed Student’s t test; mean ± SEM.
Cell Reports  , DOI: ( /j.celrep )
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