aar 003  (Alomone Labs)

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. α 1 +/- and α 1 +/+ platelets in PRP were pooled from five mice from the same strain, and platelet concentration was adjusted to 2.5E+08/mL with PPP. The PRP was divided into 4 equal parts, repleted with MgCl 2 /CaCl 2 to a final concentration of 1 mM, and then treated with 2.5 µM ADP for the indicated periods with rotation. The reaction was stopped by adding an EDTA/PGE1 cocktail containing 500 ng/mL PGE1 and 2 mM EDTA. Platelets were pelleted by a quick spin at 13,000 rpm for 15 seconds, lysed in RIPA buffer, and used for western blot assay of AKT activation. GAPDH was blotted as a loading control. The blot represents two repeats. B. WT platelets pooled from 6 mice were lysed and used for IP of NKA α1 (with ab2872 antibody, 2 µg antibody/1 mg total protein), and then IB for P2Y12 was conducted. IP was also conducted with #1 WT sample pool with normal mouse IgG (2 µg normal IgG/1 mg total protein) as control. C. Platelet lysates prepared from α1 +/- and α 1 +/+ mice were subjected to the Blue-Native PAGE assay. The membrane was blotted for α1 first, stripped, and re-blotted for P2Y12. The image represents two repeats. D. COS-7 cells were transiently transfected with P2Y12-tango plasmid (p-hP2Y12, addgene, #66471), or pEYFP-N1-A2BR (p-hA2BR, addgene, #37202) for 36 h and then cell lysates were used for IP α1 (with ab2872) and immunoblotted for P2Y12. The membrane was stripped and re-blotted for α1 and A2BR (no band was detected in the IP and data was not shown).

Article Snippet: Antibodies to P2Y12 (AGP-098) and A2BR (AAR-003) were purchased from Alomone Lab. Antibodies to NKAα1 subunit (ab2872, ab7671, and ab76020) and P2Y12 (ab184411, ab183066) were purchased from abcam (Waltham, MA).

Techniques: Concentration Assay, Western Blot, Activation Assay, Control, Blue Native PAGE, Membrane, Transfection, Plasmid Preparation

Journal: Purinergic Signalling

Article Title: Blockage of the adenosine A 2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

doi: 10.1007/s11302-023-09952-z

Figure Lengend Snippet: Cardiac fibroblasts (CFs) isolated from MCT-treated rats overexpress the adenosine A 2B receptor subtype. Shown are representative immunofluorescence confocal microscopy images of RV myocardium sections ( A ) and isolated CFs from the RV and LV myocardium ( B ) of CTRL and MCT-treated rats stained against A 2B AR (green) and vimentin (red). Nuclei are stained in blue with DAPI. Micrographs are representative of at least five different individuals and were obtained with a laser-scanning confocal microscope using the same acquisition settings. Scale bar: 30 μm. In C , shown are representative immunoblots to document the relative amounts of the A 2B AR in CFs isolated from the RV myocardium of CTRL and MCT-treated rats allowed to grow in culture for 28 days; gels were loaded with 150 μg protein amounts. Two protein species were recognized by the A 2B AR antibody (#AAR-003, Alomone Inc., Jerusalem, Israel) corresponding to a peptide near the predicted molecular weight of the A 2B AR (~37 kDa) and a higher molecular mass (~45 kDa) isotype. Please note that the naturally occurring A 2B AR isotype is highly enriched in CFs isolated from the RV myocardium of MCT-treated rats compared to their CTRL littermates. Both bands fully disappeared after pre-adsorption of the A 2B AR primary antibody with a 10-fold molar excess of the A 2B AR blocking peptide (#BLP-AR003, Alomone Inc., Jerusalem, Israel) corresponding to amino acid residues 147–166 of the human A 2B AR second extracellular loop (negative control). The rat urinary bladder (RB) was used as a positive control for the A 2B AR. β-Tubulin (55 kDa) was used as a reference protein. Each bar represents pooled data from three different individuals; three replicas were performed in each experiment. The vertical bars represent SEM. * p < 0.05 (Student’s unpaired t -test) represents significant differences compared to the CTRL group

Article Snippet: Cultured cells were fixed in 4% PFA in PBS for 10 min, washed 3 times in PBS (10 min each), and, subsequently, incubated with blocking buffer I (10% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) for 1 h. Primary antibodies diluted in blocking buffer II (5% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) were applied [mouse anti-porcine vimentin 1:250 (DAKO); goat anti-human DDR2 1:25 (Santa Cruz); mouse anti-human α-smooth muscle actin (SMA)-FITC 1:250 (Sigma); rabbit anti-human A 2B AR (2nd extracellular loop, 36 kDa) 1:50 (#AAR-003, Alomone Labs)] and the slides incubated overnight at 4 °C.

Techniques: Isolation, Immunofluorescence, Confocal Microscopy, Staining, Microscopy, Western Blot, Molecular Weight, Adsorption, Blocking Assay, Negative Control, Positive Control

Journal: Purinergic Signalling

Article Title: Blockage of the adenosine A 2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

doi: 10.1007/s11302-023-09952-z

Figure Lengend Snippet: Selective A 2B receptor blockage with PSB603 (100 nM) attenuates NECA-induced overgrowth of cardiac fibroblasts (CFs) from the RV of MCT-treated rats. NECA (10 μM) with or without PSB603 (100 nM) was incorporated in culture media throughout the whole assay. The ordinates represent NECA- and/or PSB603-induced changes in cell growth (MTT assay, A ) and type I collagen production (Sirius Red assay, B ) compared to the control situation using the same cell batch in the absence of test drugs at culture days 7, 14, 21, and 28. Zero represents the similarity between the two values (drug vs. control); positive and negative values represent facilitation or inhibition of either cell growth or type I collagen production relative to control data obtained at the same time points. Each bar represents pooled data from 5 (CTRL) and 6 (MCT) animals; 3 to 4 replicas were performed for each experiment. The vertical bars represent SEM. * p < 0.05 (ANOVA, one-way analysis of variance) represents significant differences from control values obtained in the absence of test drugs; # p < 0.05 (ANOVA, one-way analysis of variance) represents significant differences compared to the effect of NECA alone

Article Snippet: Cultured cells were fixed in 4% PFA in PBS for 10 min, washed 3 times in PBS (10 min each), and, subsequently, incubated with blocking buffer I (10% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) for 1 h. Primary antibodies diluted in blocking buffer II (5% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) were applied [mouse anti-porcine vimentin 1:250 (DAKO); goat anti-human DDR2 1:25 (Santa Cruz); mouse anti-human α-smooth muscle actin (SMA)-FITC 1:250 (Sigma); rabbit anti-human A 2B AR (2nd extracellular loop, 36 kDa) 1:50 (#AAR-003, Alomone Labs)] and the slides incubated overnight at 4 °C.

Techniques: MTT Assay, Control, Inhibition