Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Primary and secondary antibodies used to stain human and rat detrusor strips

Article Snippet: Observations were performed and analysed with a laser‐scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan; RRID:SCR_016840) and analysed with the Fluoview FV10‐ASW software, RRID:SCR_014215. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code/identifier Host Dilution Supplier Primary antibodies Anti‐β 2 receptor AAR‐016/RRID:AB_2039718 Rabbit 1:50 Alomone Anti‐β 3 receptor AAR‐017/RRID:AB_2039720 Rabbit 1:50 Alomone Anti‐β 3 receptor MC‐4198/RRID:AB_590525 Rabbit 1:50 MBL International Anti‐EPAC1 ab21236/RRID:AB_2177464 Rabbit 1:75 Abcam Anti‐EPAC1 SC‐28366/RRID:AB_627521 Mouse 1:100 Santa Cruz Anti‐EPAC2 #4156/RRID:AB_1904112 Mouse 1:50 Cell Signaling Tech Anti‐ENT1 ANT‐051/RRID:AB_2341015 Rabbit 1:50 Alomone Secondary antibodies Alexa Fluor 488 anti‐rabbit A‐21206/RRID:AB_2535792 Donkey 1:1,000 Molecular Probes Alexa Fluor 633 anti‐mouse A‐21050/RRID:AB_2535718 Goat 1:1,000 Molecular Probes Open in a separate window Primary and secondary antibodies used to stain human and rat detrusor strips

Techniques: Staining

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Primary and secondary antibodies used to stain human and rat detrusor strips

Article Snippet: The membranes were, then, blocked in Tris‐buffered saline (in mM: Tris–HCl 10 [pH 7.6], NaCl 150) containing Tween 20 0.05% and BSA 5% and, subsequently, incubated either with the following primary antibodies: rabbit anti‐β 2 (AAR‐016; 1:200) and anti‐β 3 (AAR‐017; 1:200) adrenoceptor primary antibodies from Alomone Labs (Jerusalem, Israel) or with rabbit anti‐EPAC1 (ab21236; 1:100; Abcam, Cambridge, UK) and mouse anti‐EPAC2 (5B1, mAb #4156; 1:500; Cell Signaling Technology, Danvers, MA, USA), in the above blocking buffer overnight at 4°C.

Techniques: Staining

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Mechanism of action of drugs used in this study

Article Snippet: The membranes were, then, blocked in Tris‐buffered saline (in mM: Tris–HCl 10 [pH 7.6], NaCl 150) containing Tween 20 0.05% and BSA 5% and, subsequently, incubated either with the following primary antibodies: rabbit anti‐β 2 (AAR‐016; 1:200) and anti‐β 3 (AAR‐017; 1:200) adrenoceptor primary antibodies from Alomone Labs (Jerusalem, Israel) or with rabbit anti‐EPAC1 (ab21236; 1:100; Abcam, Cambridge, UK) and mouse anti‐EPAC2 (5B1, mAb #4156; 1:500; Cell Signaling Technology, Danvers, MA, USA), in the above blocking buffer overnight at 4°C.

Techniques:

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Immunolocalization of (a) β3‐ and (b) β2‐adrenoceptors, (c) EPAC1 and (d) EPAC2 isoforms, and (e) ENT1 in transverse sections of the human (left hand‐side panels) and rat (right hand‐side panels) detrusor by confocal microscopy. β3‐Adrenoceptor and ENT1 immunoreactivities follow a plasma membrane staining pattern, whereas EPAC1 is compatible with a diffuse cytoplasmic distribution. Two distinct β3 (AAR‐017 and MC‐4198 raised in rabbits) and EPAC (ab21236 and SC‐28366 raised in rabbits and mice, respectively) antibodies were used as indicated. (f) Micrographs show that β3‐adrenoceptors (green) and EPAC (red) co‐localize in human detrusor, as identified by the yellow staining in the merge image shown in the right hand‐side image. Images are representative of four different individuals. Differential interference contrast (DIC) images are shown for comparison. Scale bar = 50 μm. Bottom panels are representative immunoblots of β2‐ and β3‐adrenoceptors (g) and EPAC1 and EPAC2 isoforms (h) in urothelium‐denuded detrusor homogenates of one male human subject (H) and one rat (R) run in parallel using the same antibodies as for the immunohistochemical staining. The highly conserved GAPDH enzyme (EC 1.2.1.12, MW~37 kDa) and β‐actin (MW~43 kDa) were used as reference proteins for β−adrenoceptors and EPAC respectively. Images are representative of three different individuals. Gels were loaded with 150 μg of protein. Host species for antibody production were rabbit (rb) and mouse (ms)

Article Snippet: Anti‐β 3 receptor , AAR‐017/RRID:AB_2039720 , Rabbit , 1:50 , Alomone.

Techniques: Confocal Microscopy, Staining, Western Blot, Immunohistochemical staining