rabbit anti a2br  (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: International Journal of Molecular Sciences

Article Title: Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells

doi: 10.3390/ijms23094585

Figure Lengend Snippet: Relation between ADORA2B transcript (coding for A2BR) and survival time for patients with OC. Kaplan–Meier plots were constructed for the ADORA2B transcript using the Kaplan–Meier plotter database (KMPdb) for ( A ) Ovarian carcinoma in general carcinoma patients (996 low expression and 439 high expression); ( B ) Serous ovarian carcinoma patients (482 low expression and 622 high expression); ( C ) Serous ovarian carcinoma patients in stages 1 + 2 (50 low expression and 49 high expression) and ( D ) Serous ovarian carcinoma patients in stages 3 + 4 (396 low expression and 605 high expression).

Article Snippet: For detection, membranes were incubated overnight at 4 °C with the following primary antibodies in 1:1000 dilution: anti-phospho p44/42 MAPK, anti-total p42/p44 MAPK, anti-E-cadherin (Cell signaling Technologies, Danvers, MA, EUA) or anti-A2BR (Alomone Labs, Jerusalem, Israel).

Techniques: Construct, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells

doi: 10.3390/ijms23094585

Figure Lengend Snippet: Ovarian carcinoma-derived SKOV-3 cells express functional A2BR receptors. ( A ) A fragment of the ADORA2B transcript was amplified by RT and PCR using specific oligonucleotides. An amplicon of 161 bp was obtained. GAPDH was used as a constitutive transcript ( left panels ). A2BR was detected by Western blot and immunoprecipitation of biotinylated membrane proteins from SKOV-3 cells; a main band of around 42 kDa was detected ( right panel ). ( B ) A2BR was labeled by immunofluorescence. The immunoreactivity was detected with a secondary antibody conjugated with AlexaFluor 488 (green signal). Nuclei were counterstained with DAPI (blue signal). ( C ) Cell cultures were stimulated with 10 μM of BAY-606583 for 1, 3, 5 or 15 min. Phosphorylated ERK (p-ERK) and total ERK (t-ERK) were detected by Western blot in the same membrane. As a positive control, UTP 100 μM was utilized. ( D ) ERK phosphorylation induced after 5 min of stimulation with BAY-606583 was prevented by the A2BR antagonist PSB-603; this effect was also inhibited by a PKA inhibitor ( E ) or by U73122, a PLC inhibitor ( F ). In the graphs, bars represent the mean ± S.E.M. of four independent experiments in duplicate. * p < 0.05, ** p < 0.01.

Article Snippet: For detection, membranes were incubated overnight at 4 °C with the following primary antibodies in 1:1000 dilution: anti-phospho p44/42 MAPK, anti-total p42/p44 MAPK, anti-E-cadherin (Cell signaling Technologies, Danvers, MA, EUA) or anti-A2BR (Alomone Labs, Jerusalem, Israel).

Techniques: Derivative Assay, Functional Assay, Amplification, Western Blot, Immunoprecipitation, Labeling, Immunofluorescence, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells

doi: 10.3390/ijms23094585

Figure Lengend Snippet: Stimulation of A2BR inhibits basal SKOV-3 cell migration but has no effect on cell viability. ( A ) Cultures of SKOV-3 cells at 50% confluence were stimulated for 24 h with BAY-606583 from 10 nM to 10 μM; then, cell viability was estimated by MTS assay. ( B ) Confluent cultures of SKOV-3 cells were stimulated for 16 h with BAY-606583 from 100 nM to 10 μM, and cell migration was estimated by scratch assay. UTP (100 μM) and Apy were used as positive and negative controls, respectively. ( C ) SKOV-3 cells were treated for 16 h with 100 μM UTP, 10 U/mL Apy or 10 μM BAY-606583; then, the actin cytoskeleton was stained with rhodamine-coupled phalloidin, and nuclei were counterstained with DAPI. In the graphs, bars represent the mean ± S.E.M. of four independent experiments in sextuplicate for A and in triplicate for B. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For detection, membranes were incubated overnight at 4 °C with the following primary antibodies in 1:1000 dilution: anti-phospho p44/42 MAPK, anti-total p42/p44 MAPK, anti-E-cadherin (Cell signaling Technologies, Danvers, MA, EUA) or anti-A2BR (Alomone Labs, Jerusalem, Israel).

Techniques: Migration, MTS Assay, Wound Healing Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells

doi: 10.3390/ijms23094585

Figure Lengend Snippet: Overexpression of A2BR inhibits basal migration of SKOV-3 cells. SKOV-3 cells were transfected with a plasmid coding for A2BR fused with yellow fluorescent protein (YFP) at the carboxy-end; another plasmid coding only for YFP was used as a control. After transfection, cells were cultured to reach confluence (48 h) and a scratch assay was performed. Pictures show the wound at the time it was made and 16 h after. In the graphs, bars represent the mean ± S.E.M. of three independent experiments. ** p < 0.05, *** p < 0.01. Scale bar = 250 µm

Article Snippet: For detection, membranes were incubated overnight at 4 °C with the following primary antibodies in 1:1000 dilution: anti-phospho p44/42 MAPK, anti-total p42/p44 MAPK, anti-E-cadherin (Cell signaling Technologies, Danvers, MA, EUA) or anti-A2BR (Alomone Labs, Jerusalem, Israel).

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Cell Culture, Wound Healing Assay

Journal: International Journal of Molecular Sciences

Article Title: Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells

doi: 10.3390/ijms23094585

Figure Lengend Snippet: Stimulation of A2BR with BAY-606583 incremented the expression and induced the relocation of E-cadherin. SKOV-3 cell cultures were stimulated for 16 h with 10 μM BAY-606583, 10 μM ADO or 100 μM UTP. E-cadherin was detected by immunofluorescence ( A ) using a specific primary antibody and a secondary one coupled to AlexaFluor 488 (green signal). Nuclei were counterstained with DAPI (blue signal) and Western blot ( B ), where the abundance of E-cadherin was expressed in relation with against β-actin as housekeeping protein. In the graph, bars represent the mean ± S.E.M. of three independent experiments, *** p < 0.01 vs. Ctrl.

Article Snippet: For detection, membranes were incubated overnight at 4 °C with the following primary antibodies in 1:1000 dilution: anti-phospho p44/42 MAPK, anti-total p42/p44 MAPK, anti-E-cadherin (Cell signaling Technologies, Danvers, MA, EUA) or anti-A2BR (Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells

doi: 10.3390/ijms23094585

Figure Lengend Snippet: Regulation of gene expression patterns by A2BR activation with BAY-606583 in SKOV-3 cells was analyzed by cDNA microarrays. SKOV-3 cell cultures were stimulated with 10 μM BAY-606583 for 16 h and total RNA was isolated; a pool of five independent cultures was hybridized against a library of 30 K genes of the complete human genome. ( A ) BAY-606583 induced broad changes in gene expression; the Venn diagram represents the set of transcripts up- or down-regulated by the agonist. ( B ) A heat-map representation of the Z-score of outstanding transcripts up- or down-regulated by BAY-606583. The cellular process determined by GO analysis is shown.

Article Snippet: For detection, membranes were incubated overnight at 4 °C with the following primary antibodies in 1:1000 dilution: anti-phospho p44/42 MAPK, anti-total p42/p44 MAPK, anti-E-cadherin (Cell signaling Technologies, Danvers, MA, EUA) or anti-A2BR (Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Activation Assay, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells

doi: 10.3390/ijms23094585

Figure Lengend Snippet: Transcripts regulated by A2BR stimulation with 10 mM of BAY for 24 h.

Article Snippet: For detection, membranes were incubated overnight at 4 °C with the following primary antibodies in 1:1000 dilution: anti-phospho p44/42 MAPK, anti-total p42/p44 MAPK, anti-E-cadherin (Cell signaling Technologies, Danvers, MA, EUA) or anti-A2BR (Alomone Labs, Jerusalem, Israel).

Techniques: Migration, Binding Assay