Journal: Cell communication and signaling : CCS

Article Title: CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in G αs -G αi coupling to the β 2 -adrenergic receptor.

doi: 10.1186/s12964-023-01358-z

Figure Lengend Snippet: Fig. 2 Chronic Epi stimulation inhibits cAMP production and activates FLSs through β2AR. A Intracellular cAMP production induced by ISO (100 nM) stimulation in normal rat FLSs that were treated with ISO (1 μM) in the presence or absence of CGP (1 μM), ICI (1 μM) or 100 nM SR overnight was detected in the FRET system. B Intracellular cAMP production in normal or CIA rat FLSs treated with Ter (10 μM), (10 μM), or ISO (1 μM) was detected in the FRET system. C The intracellular cAMP concentration in FLSs from normal or CIA rats treated with Ter or ISO was determined by a kit. D The effect of knocking down β2AR on FLS viability was detected by a CCK-8 assay. E, F The effects of knocking down β2AR on FLS migration were determined by a Transwell assay, and the data were analysed. Scale bar, 100 μm. G, H The effects of knocking down β2AR on FLS invasion were determined by a Transwell assay, and the data were analysed. Scale bar, 100 μm. The data are presented as the means ± SEMs; *p < 0.05, **p < 0.01, ***p < 0.001; n = 5 animals per group

Article Snippet: The precleared protein was then incubated with 10 μl of Protein A/G PLUS-Agarose beads preincubated with the anti-β2AR antibody (Catalogue # sc-570, Santa Cruz, CA, USA) overnight at 4 °C with rotation.

Techniques: Concentration Assay, CCK-8 Assay, Migration, Transwell Assay

Journal: Cell communication and signaling : CCS

Article Title: CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in G αs -G αi coupling to the β 2 -adrenergic receptor.

doi: 10.1186/s12964-023-01358-z

Figure Lengend Snippet: Fig. 7 Graphical abstract. In normal synovial tissue, β-adrenergic receptors on FLSs activate adenylyl cyclase mainly by coupling with Gαs, thereby maintaining a physiological intracellular cAMP level. In the inflammatory environment, increased Epi leads to a GRK2-mediated switch in Gαs-Gαi coupling to β2AR on FLSs and a decrease in intracellular cAMP production and subsequently promotes FLS proliferation, migration and invasion, resulting in RA. The novel GRK2 inhibitor CP-25 inhibits the hyperactivation of rheumatic synoviocytes and alleviates CIA through restoration of Gαs coupling to β2AR and maintenance of the β2AR response in FLSs

Article Snippet: The precleared protein was then incubated with 10 μl of Protein A/G PLUS-Agarose beads preincubated with the anti-β2AR antibody (Catalogue # sc-570, Santa Cruz, CA, USA) overnight at 4 °C with rotation.

Techniques: Migration

Journal: Disease Models & Mechanisms

Article Title: Chronic psychosocial stress disturbs long-bone growth in adolescent mice

doi: 10.1242/dmm.030916

Figure Lengend Snippet: Sympathetic signaling in the femur and the adrenal gland following 19 days of chronic subordinate colony housing (CSC). (A) Immunofluorescence staining for tyrosine hydroxylase (TH) at the growth plate (GP) and (B) the metaphyseal region (C, cortex) of the femur. (C) β2-adrenergic receptor (β2AR) staining (arrows) at the GP and (D) the metaphyseal region of the femur. Scale bars: 50 µm, n =4-6. (E) Western blot analysis of TH in tibia lysates; β-actin was used as the reference protein. (F) Relative gene expression of TH analyzed by qPCR from distal tibia homogenates. Values were normalized to B2M . (G,H) Western blot analysis of TH in adrenal gland lysates. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference protein. SHC, single-housed control. Data are displayed as individual dot plots with median (red)±range (black). *0.05≥ P ≥0.01.

Article Snippet: Immunofluorescence staining for TH and β2AR was performed using the following antibodies: rabbit anti-mouse β2AR antibody (sc-569, Santa Cruz), rabbit-anti mouse TH antibody (AB152, Millipore), goat-anti rabbit IgG-biotin (sc-3840, Santa Cruz) and FITC-streptavidin (40201, BioLegend).

Techniques: Immunofluorescence, Staining, Western Blot, Gene Expression, Control