anti β2 ar antibody  (Alomone Labs)

Journal: Current Issues in Molecular Biology

Article Title: Catecholamines Attenuate LPS-Induced Inflammation through β2 Adrenergic Receptor Activation- and PKA Phosphorylation-Mediated TLR4 Downregulation in Macrophages

doi: 10.3390/cimb46100675

Figure Lengend Snippet: Concentration-dependent effects of adrenaline (Ad), noradrenaline (Nad), dopamine (DA), and acetylcholine (ACh) on LPS-induced TNFα production in RAW264.7 cells. Cells were treated with varying concentrations of ( a ) Ad (0.01–10 μM), ( b ) Nad (0.01–10 μM), ( c ) DA (0.1–100 μM), and ( d ) ACh (0.01–10 μM), in the presence or absence of 1 μg/mL LPS for 2 h. TNFα expression was assessed at both the mRNA level (upper panel) via RT-qPCR and the protein level (lower panel) via immunoblotting. Data from qPCR analyses are expressed as mean ± SE ( n = 3). *** p < 0.001 vs. without any treatment; ## p < 0.01 and ### p < 0.001 vs. with LPS treatment. For the immunoblotting, one representative result from three independent experiments is shown. GAPDH was used as a loading control.

Article Snippet: In addition, some membranes were probed with an antibody against β2-AR (1:500, sc-81577, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Concentration Assay, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Current Issues in Molecular Biology

Article Title: Catecholamines Attenuate LPS-Induced Inflammation through β2 Adrenergic Receptor Activation- and PKA Phosphorylation-Mediated TLR4 Downregulation in Macrophages

doi: 10.3390/cimb46100675

Figure Lengend Snippet: Roles of the β2 adrenergic receptor (β2-AR) in the effects of Ad, Nad, and DA on TNFα expression in LPS-treated RAW264.7 cells. ( a ) AR expression in RAW 264.7 cells. Effects of phentolamine, propranolol, metoprolol, or ICI 118,551 on TNFα expression in cells treated with LPS with or without ( b ) Ad, ( c ) Nad, and ( d ) DA. ( e ) Cells were also treated with LPS with or without metaramine, isoproterenol, dobutamine, or fenoterol. Data from qPCR analyses are expressed as means ± SE ( n = 3). ** p < 0.01, and *** p < 0.001 vs. without any treatment; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. with LPS treatment. For the immunoblotting, one representative result from three independent experiments is shown. GAPDH was used as a loading control.

Article Snippet: In addition, some membranes were probed with an antibody against β2-AR (1:500, sc-81577, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Current Issues in Molecular Biology

Article Title: Catecholamines Attenuate LPS-Induced Inflammation through β2 Adrenergic Receptor Activation- and PKA Phosphorylation-Mediated TLR4 Downregulation in Macrophages

doi: 10.3390/cimb46100675

Figure Lengend Snippet: Effects of β2-AR knockdown or overexpression on the expression of TNFα in LPS-treated RAW264.7 cells treated with Ad. Cells were transfected with the specific siRNA targeting β2-AR (β2-AR-siRNA) and control siRNA (Con-siRNA), and plasmid overexpressing β2-AR (pcDNA-β2-AR) and its control (pcDNA), respectively. ( a , c ) β2-AR expression was then examined via immunoblotting. ( b , d ) Transfected cells were treated with LPS with or without Ad for 2 h, and TNFα protein levels were determined (upper panel). One representative result from three independent experiments is shown. GAPDH was used as a loading control. The band density was quantitatively analyzed using ImageJ software. The results (lower panel in ( b , d )) are expressed as mean ± SE ( n = 3). *** p < 0.001; # p < 0.05 and ### p < 0.001; ††† p < 0.001.

Article Snippet: In addition, some membranes were probed with an antibody against β2-AR (1:500, sc-81577, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Knockdown, Over Expression, Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Software

Journal: Current Issues in Molecular Biology

Article Title: Catecholamines Attenuate LPS-Induced Inflammation through β2 Adrenergic Receptor Activation- and PKA Phosphorylation-Mediated TLR4 Downregulation in Macrophages

doi: 10.3390/cimb46100675

Figure Lengend Snippet: Roles of β2-AR activation in PKA phosphorylation (p-PKA) and TLR4 expression in RAW264.7 cells. ( a , b ) Cells were transfected as described above, and TLR4 protein levels were assessed. After transfection with pcDNA or pcDNA-β2-AR for 8 h, H89 was added and incubated for 10 h. Ad was then added and incubated for 2 h. PKA, p-PKA, and TLR4 protein levels were detected (upper panel in ( c , d )). One representative result from three independent experiments is shown. GAPDH was used as a loading control. Band density was quantitatively analyzed using ImageJ software, and the results (lower panel) are expressed as means ± SE ( n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001; # p < 0.05; † p < 0.05.

Article Snippet: In addition, some membranes were probed with an antibody against β2-AR (1:500, sc-81577, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Transfection, Incubation, Control, Software

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 membrane was blocked in Tris-buffered saline containing 0.05% Tween 20 (TBST) and 5% nonfat milk at 37 °C for 2 h, followed by incubation with a primary antibody against β1AR (1:1000, Catalogue # PA1-049, Thermo Fisher Scientific, Waltham, MA, USA), β2AR (1:600, Catalogue # sc-570, Santa Cruz Biotechnology, CA, USA), β3AR (1:500, Catalogue # YT0363, Immunoway, TX, USA), Gαs (1:500, Catalogue # sc-823, Santa Cruz Biotechnology, CA, USA), Gαi (1:500, Catalogue # sc-391, Santa Cruz Biotechnology, CA, USA), or GAPDH (1:5000, Catalogue # AF0911, Affinity Biosciences, Changzhou, China) overnight at 4 °C.

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 membrane was blocked in Tris-buffered saline containing 0.05% Tween 20 (TBST) and 5% nonfat milk at 37 °C for 2 h, followed by incubation with a primary antibody against β1AR (1:1000, Catalogue # PA1-049, Thermo Fisher Scientific, Waltham, MA, USA), β2AR (1:600, Catalogue # sc-570, Santa Cruz Biotechnology, CA, USA), β3AR (1:500, Catalogue # YT0363, Immunoway, TX, USA), Gαs (1:500, Catalogue # sc-823, Santa Cruz Biotechnology, CA, USA), Gαi (1:500, Catalogue # sc-391, Santa Cruz Biotechnology, CA, USA), or GAPDH (1:5000, Catalogue # AF0911, Affinity Biosciences, Changzhou, China) overnight at 4 °C.

Techniques: Migration