Journal: Scientific Reports

Article Title: New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D 2 receptor regulation and signaling

doi: 10.1038/s41598-021-87417-2

Figure Lengend Snippet: Agonist-induced GRK2 recruitment, Ser 317 /Thr 318 phosphorylation and β-arrestin2 recruitment. ( A ). HEK293 cells were transfected GRK2-Venus and hD 2L R-NLuc. GRK2 recruitment was measured by BRET 10 min after agonist addition at 37 °C. Data is presented as the increase in BRET ratio normalized to vehicle (0%) and the maximal effect of dopamine (100%). Data represents the mean ± SEM of 5 separate experiments performed in duplicate. ( B ) HA-hD 2L R expressing HEK293 cells were either stimulated with vehicle (solvent) or 10 μM of quinpirole (quin), dopamine (dop), pergolide (perg), ropinirole (rop), apomorphine (apo), cabergoline (cabergo), bromocriptine (bromo), terguride (terg), roxindole (roxin), aripiprazole (arip), MLS1547 (MLS) or UNC9994 (UNC) for 10 min at 37 °C. Lysates were immunoblotted with antibody to pSer 317 /Thr 318 [5102]. Blots were stripped and reprobed for D 2 R [5106] to confirm equal loading of the gel. Blots are representative, n = 3. ( C ) These Western blots were analyzed using densitometry to yield relative pSer 317 /pThr 318 signals normalized to the corresponding total D 2 R signal. Data represents the mean ± S.E.M. of three separate experiments. ( D ) Agonist-induced β-arrestin2 recruitment to the D 2 R. HEK293 cells were transfected with hD 2L R-NLuc, GRK2 and YFP-β-arrestin2. β-arrestin2 recruitment was measured by BRET 10 min after agonist addition at 37 °C. Data is presented as the increase in BRET ratio normalized to vehicle (0%) and the maximal effect of dopamine (100%). Data represents the mean ± SEM of 5 separate experiments performed in duplicate. ( E – G ) Correlation of the effect of a saturating concentration of each agonist (10 μM) to stimulate Ser 317 /Thr 318 phosphorylation maximal with the maximal effects of the various agonists in assays measuring GRK2 recruitment and β-arrestin2 recruitment determined. DA is marked in blue. The relationship between two variables was assessed using a two-tailed Spearman’s rank correlation allowing the calculation of the correlation coefficient, r s . A P value of 0.05 was used as the cut-off for statistical significance and relationships depicted as trend lines.

Article Snippet: Apomorphine hydrochloride (2073), MLS1547 (6171), ropinirole (3680), quinpirole hydrochloride (1061), dopamine hydrochloride (3548), cabergoline (2664), bromocriptine mesylate (0427), forskolin (1099), SCH23390 (0925), PTX (3097), haloperidol hydrochloride (0931), L-741,626 (1003) and roxindole (1559) were obtained from Tocris.

Techniques: Phospho-proteomics, Transfection, Expressing, Solvent, Western Blot, Concentration Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D 2 receptor regulation and signaling

doi: 10.1038/s41598-021-87417-2

Figure Lengend Snippet: Potency (pEC 50 ) and maximal effect (E max ) estimates for agonists activating D 2 R regulatory pathways.

Article Snippet: Apomorphine hydrochloride (2073), MLS1547 (6171), ropinirole (3680), quinpirole hydrochloride (1061), dopamine hydrochloride (3548), cabergoline (2664), bromocriptine mesylate (0427), forskolin (1099), SCH23390 (0925), PTX (3097), haloperidol hydrochloride (0931), L-741,626 (1003) and roxindole (1559) were obtained from Tocris.

Techniques:

Journal: Scientific Reports

Article Title: New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D 2 receptor regulation and signaling

doi: 10.1038/s41598-021-87417-2

Figure Lengend Snippet: Concentration-dependent agonist-induced Ser 317 /Thr 318 phosphorylation. ( A ) HA-hD 2 R expressing HEK293 cells were either stimulated with vehicle (solvent) or quinpirole, dopamine, pergolide, ropinirole, apomorphine, cabergoline, bromocriptine, terguride, roxindole, aripiprazole, MLS1547 or UNC9994 at concentrations ranging from 10 –9 to 10 –5 M for 10 min at 37 °C. Lysates were immunoblotted with antibody to pSer 317 /Thr 318 [5102]. Blots were stripped and reprobed for D 2 R [5106] to confirm equal loading of the gel. Blots are representative, n = 3. ( B ) Densitometry analysis of Western blots. pSer 317 /pThr 318 signals were normalized to the total D 2 R signal and expressed as a percentage of the signal detected when cells were stimulated with 10 μM dopamine (Fig. 4). These data are fitted to a three parameter concentration response curve (Eq. ).

Article Snippet: Apomorphine hydrochloride (2073), MLS1547 (6171), ropinirole (3680), quinpirole hydrochloride (1061), dopamine hydrochloride (3548), cabergoline (2664), bromocriptine mesylate (0427), forskolin (1099), SCH23390 (0925), PTX (3097), haloperidol hydrochloride (0931), L-741,626 (1003) and roxindole (1559) were obtained from Tocris.

Techniques: Concentration Assay, Phospho-proteomics, Expressing, Solvent, Western Blot

Journal: Scientific Reports

Article Title: New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D 2 receptor regulation and signaling

doi: 10.1038/s41598-021-87417-2

Figure Lengend Snippet: Potency (pEC 50 ) and maximal effect (E max ) estimates for agonists activating D 2 R G protein signalling pathways.

Article Snippet: Apomorphine hydrochloride (2073), MLS1547 (6171), ropinirole (3680), quinpirole hydrochloride (1061), dopamine hydrochloride (3548), cabergoline (2664), bromocriptine mesylate (0427), forskolin (1099), SCH23390 (0925), PTX (3097), haloperidol hydrochloride (0931), L-741,626 (1003) and roxindole (1559) were obtained from Tocris.

Techniques:

Journal: Frontiers in Immunology

Article Title: Regulatory B Cells Dysregulated T Cell Function in an IL-35-Dependent Way in Patients With Chronic Hepatitis B

doi: 10.3389/fimmu.2021.653198

Figure Lengend Snippet: Suppression effect on effector cells (ECs). B cells were isolated from PBMC using CD19+ microbead and stimulated with CD40L/CpG/LPS with or without HBVcore peptide. Stimulated B cells were then co-cultured with autologous CD19-depleted PBMCs at the ratio of 2:1 in anti-CD3/CD28 coated plate for 48hours. As a control, CD19-depleted PBMCs were also cultured alone without B cells (CD19-depleted PBMCs non-activated and CD19-depleted PBMCs activated). (A) Suppression effect on IFN-ɤ-producing CD4+T cells; (B) Suppression on IL-4-producing CD4+T cells; (C) Suppression effect on IL-17A-producing CD4+T cells; (D) Suppression on IFN-ɤ-producing CD8+T cells; (E) Suppression on IL-4-producing CD8+T cells. Non-parametric Kruskal-Wallis ANOVA test and Tukey’s Multiple Comparison test was used to analysis the data. P value<;0.05 was considered statistically significant.

Article Snippet: For Breg subset and B10 or IL-35+B cell detection, PBMCs (2 × 10 6 ) were stimulated with CD40L (5 μg/mL, Purified Anti-Human CD154 (CD40L), Tonbo Biosciences, USA) plus CpG-ODN (1.5 μM, TLR9 Agonist-Stimulatory Class B tlrl-2006, InvivoGen) and lipopolysaccharide (LPS, 1 μg/mL, eBioscience) for 48 h. Furthermore, cells were activated for another 4 h at 37°C in 5% CO 2 with 50 ng/mL phorbol myristate acetate, 1 mmol/L ionomycin (both from Sigma, St. Louis, MO, USA), and 10 mg/mL brefeldin A (Tocris Cookson, Bristol, UK) in complete RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA).

Techniques: Isolation, Cell Culture, Control, Comparison