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G protein-coupled receptor 35 (GPR35) activation reduces c-Src and PI3K-Akt signaling pathways in mature osteoclasts. Quantification of phosphorylated (A) c-Src (pSrc), (B) Akt1/2/3 (pAkt), and (C) p38 (p-p38) measured by AlphaLISA in osteoclasts exposed to vehicle or GPR35 agonists with or without GPR35 antagonist ML145. (D-E) Quantification of (D) c-Src and (E) Akt1/2/3 phosphorylation in mature osteoclasts transfected with scrambled or GPR35 siRNA exposed to vehicle or GPR35 agonist, TC-G 1001. Data in all panels was normalized to GAPDH as a housekeeper control. Each point represents an independent donor in all panels. The gray line denotes mean in panels a, B, D and E and median in panel C. Statistical analyses were performed by one-way ANOVA with Holm-Šídák’s multiple comparisons test in panels A, B, D, and E and Kruskal–Wallis with Dunn’s multiple comparisons test in C. **** p < .0001, *** p < .001, ** p < .01, * p < .05.

Journal: JBMR Plus

Article Title: G protein-coupled receptor 35 stimulation reduces osteoclast activity in primary human bone cells

doi: 10.1093/jbmrpl/ziaf131

Figure Lengend Snippet: G protein-coupled receptor 35 (GPR35) activation reduces c-Src and PI3K-Akt signaling pathways in mature osteoclasts. Quantification of phosphorylated (A) c-Src (pSrc), (B) Akt1/2/3 (pAkt), and (C) p38 (p-p38) measured by AlphaLISA in osteoclasts exposed to vehicle or GPR35 agonists with or without GPR35 antagonist ML145. (D-E) Quantification of (D) c-Src and (E) Akt1/2/3 phosphorylation in mature osteoclasts transfected with scrambled or GPR35 siRNA exposed to vehicle or GPR35 agonist, TC-G 1001. Data in all panels was normalized to GAPDH as a housekeeper control. Each point represents an independent donor in all panels. The gray line denotes mean in panels a, B, D and E and median in panel C. Statistical analyses were performed by one-way ANOVA with Holm-Šídák’s multiple comparisons test in panels A, B, D, and E and Kruskal–Wallis with Dunn’s multiple comparisons test in C. **** p < .0001, *** p < .001, ** p < .01, * p < .05.

Article Snippet: AlphaLISA readings were made on a PHERAstar FS (BMG Labtech, Aylesbury, United Kingdom) plate reader, and values for phosphorylated proteins normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) values.

Techniques: Activation Assay, Protein-Protein interactions, Phospho-proteomics, Transfection, Control

G protein-coupled receptor 35 (GPR35) mediated effects on bone resorption and signaling are maintained in osteoclast-osteoblast co-cultures. (A) Quantification of toluidine blue-stained resorption pits and trenches formed on bone slices by osteoclast-osteoblast co-cultures exposed to vehicle, TC-G 1001, or TC-G 1001 and the ML145 antagonist (B) quantification of tartrate-resistant acid phosphatase enzyme activity in conditioned media from osteoclast-osteoblast co-cultures exposed to GPR35 agonists or agonists with antagonist. (C-F) quantification of (C) phosphorylated c-Src, (D) phosphorylated Akt1/2/3, (E) phosphorylated NFκB, (F) phosphorylated cAMP response element-binding protein in osteoclast-osteoblast co-cultures exposed to GPR35 agonists or agonists with antagonist, measured by AlphaLISA. Each point represents an independent donor. The gray line denotes mean. Statistical analyses were performed by one-way ANOVA with Holm-Šídák’s multiple comparisons test for A-B and with Dunnett’s multiple comparisons test for C-F. **** p < .0001, *** p < .001, ** p < .01, * p < .05.

Journal: JBMR Plus

Article Title: G protein-coupled receptor 35 stimulation reduces osteoclast activity in primary human bone cells

doi: 10.1093/jbmrpl/ziaf131

Figure Lengend Snippet: G protein-coupled receptor 35 (GPR35) mediated effects on bone resorption and signaling are maintained in osteoclast-osteoblast co-cultures. (A) Quantification of toluidine blue-stained resorption pits and trenches formed on bone slices by osteoclast-osteoblast co-cultures exposed to vehicle, TC-G 1001, or TC-G 1001 and the ML145 antagonist (B) quantification of tartrate-resistant acid phosphatase enzyme activity in conditioned media from osteoclast-osteoblast co-cultures exposed to GPR35 agonists or agonists with antagonist. (C-F) quantification of (C) phosphorylated c-Src, (D) phosphorylated Akt1/2/3, (E) phosphorylated NFκB, (F) phosphorylated cAMP response element-binding protein in osteoclast-osteoblast co-cultures exposed to GPR35 agonists or agonists with antagonist, measured by AlphaLISA. Each point represents an independent donor. The gray line denotes mean. Statistical analyses were performed by one-way ANOVA with Holm-Šídák’s multiple comparisons test for A-B and with Dunnett’s multiple comparisons test for C-F. **** p < .0001, *** p < .001, ** p < .01, * p < .05.

Article Snippet: AlphaLISA readings were made on a PHERAstar FS (BMG Labtech, Aylesbury, United Kingdom) plate reader, and values for phosphorylated proteins normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) values.

Techniques: Staining, Activity Assay, Binding Assay