Ding sites in control individuals. Kd and Bmax RG7800MedChemExpress RG7800 values were 25 ?1.3 nmol
Ding sites in control individuals. Kd and Bmax values were 25 ?1.3 nmol/l and 35 ?2.4 fmol/mg protein, respectively (Fig. 2). Competition experiments using [3H]CGS21680 in combination with a variety of A2A ligands revealed a pharmacological profile typical for A2A ARs (R-PIA [R-N6-phenylisopropyladenosine] > teofilline > NECA > SCH58261; data not shown). Scatchard analysis for SSc neutrophils revealed no significant differences in Kd and Bmax between patients (mean values: Kd = 23 ?1.8 nmol/l, Bmax = 40 ?3.2 fmol/mg protein) and healthy control individuals (P > 0.05; Fig. 2), suggesting that no alteration in A2A binding sites occurs in SSc. In agreement with this, densitometric analysis of immunoblots showed no significant changes in A2A AR immunoreactive bands in SSc neutrophils relative to controls (optical density: 0.11 ?0.03 for patients versus 0.15 ?0.02 for controls). A2B AR binding sites were identified using [3H]NECA as radioligand in the presence of 50 nmol/l CPA and 100 nmol/l SCH58261, to prevent nonspecific binding to A1 and A2A AR subtypes. We performed competition experimentsRNeutrophils were homogenized in buffer solution containing 10 mmol/l Hepes, 1 mmol/l EGTA and 10 mmol/l NaCl2, and then centrifuged at 46,500 g for 20 min at 4 . Pellets were resuspended in 10 volumes of 10 mmol/l Hepes, containing protease inhibitors (200 /ml bacitracine and 160 /ml benzamidine), incubated for 30 min at 30 with 2 U/ml ADA, and centrifuged. Adenylyl cyclase (AC) activity was measured as described by Salomon [22] and Johnson and Salomon [23], with some modifications. NECA-mediated stimulation of AC activity was assessed by incubating aliquots of membranes with increasing NECA concentrations from 0.01 nmol/l to 10 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27324125 ol/l. The reaction was started by adding membrane aliquots (10?0 proteins/tube), conducted for 15 min at 24 , and then stopped by transferring samples on ice and adding 500 ice-cold stop solution (120 mmol/l zinc acetate, 144 mmol/ l Na2CO3). The stop solution contained [3H]cAMP (10,000?5,000 cpm/sample) to monitor column recovery. Newly formed ZnCO3 allowed precipitation of residual ATP, discarded through centrifugation at 2700 g for 8 min. Supernatants containing both [32P]-cAMP and [3H]cAMP were further purified by double-step Dowex-Alumina chromatography and counted by means of a -counter (Packard Tricarb 1600; Perkin Elmer, Wellesley, MA, USA).Arthritis Research TherapyVol 7 NoBazzichi et al.FigureFigureRepresentative Scatchard plot of [3H]CGS21680 saturation binding data data. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25636517 Empty circles indicate neutrophil membranes from healthy volunteers (affinity constant [Kd] = 25 ?1.3 nmol/l; maximum number of binding sites [Bmax] = 35 ?2.4 fmol/mg); filled circles indicate neutrophil membranes from systemic sclerosis (SSc) patients overall (Kd = 23 ?1.8 nmol/l; Bmax = 40 ?3.2 fmol/mg). Assays were performed in triplicate.systemic sclerosis (SSc) neutrophils and controls Immunoblotting analysis of A2A and A2B adenosine receptors (ARs) from systemic sclerosis (SSc) neutrophils and controls. Cells obtained from 26 healthy volunteers and 26 SSc patients were lysed as described in the Methods section. Equal amounts of protein (50 ) were separated on polyacrylamide gel, blotted and probed with 0.1 /ml rabbit antihuman A2A AR or A2B AR antibodies. Immunoreactive bands were visualized according to electrogenerated chemiluminescence protocol. A2A and A2B AR antibodies recognized immunoreactive bands of 45 kDa and 50 kDa, res.