Journal: Current Issues in Molecular Biology

Article Title: Levistolide A Alleviates Myocardial Ischemia–Reperfusion Injury Partly by Improving Calcium Homeostasis via the ADORA2B/cAMP/PKA/PLB/SERCA2α Signaling Axis

doi: 10.3390/cimb48020125

Figure Lengend Snippet: The key role of ADORA2B in the calcium signaling pathway and its molecular docking results with LA. ( A ) Schematic representation of the central position of ADORA2B in the calcium signaling pathway; ( B ) Molecular docking binding mode of LA with the ADORA2B protein.

Article Snippet: The antibodies used in this study are as follows: adenosine A2B receptor (ADORA2B) (37KDa) antibody (bs-5900R) was purchased from Bioss Biotechnology Co., Ltd. (Beijing, China); phospholamban (PLB) (36KDa) antibody (A01395-1) was purchased from Boster Biological Engineering Co., Ltd. (Wuhan, China); phosphorylated phospholamban-Ser16 (p-PLB-Ser16) (36KDa) antibody (AP0907) was purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China); SERCA2α (140KDa) antibody (9580T) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); and α-Tubulin (55 KDa) antibody (66031-1-Ig) was purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Binding Assay

Journal: Current Issues in Molecular Biology

Article Title: Levistolide A Alleviates Myocardial Ischemia–Reperfusion Injury Partly by Improving Calcium Homeostasis via the ADORA2B/cAMP/PKA/PLB/SERCA2α Signaling Axis

doi: 10.3390/cimb48020125

Figure Lengend Snippet: Protein expression levels of ADORA2B, p-PLB, PLB, and SERCA2α in different groups of H9C2 cells. ( A ) Representative Western blot bands of target proteins in each group; ( B ) Relative protein expression level of ADORA2B; ( C ) Relative protein expression level of p-PLB; ( D ) Relative protein expression level of SERCA2α. Compared to the control group, ## p < 0.01. Compared to the H/R group, ** p < 0.01. Compared to the LA + H/R group, Δ p < 0.05, ΔΔ p < 0.01.

Article Snippet: The antibodies used in this study are as follows: adenosine A2B receptor (ADORA2B) (37KDa) antibody (bs-5900R) was purchased from Bioss Biotechnology Co., Ltd. (Beijing, China); phospholamban (PLB) (36KDa) antibody (A01395-1) was purchased from Boster Biological Engineering Co., Ltd. (Wuhan, China); phosphorylated phospholamban-Ser16 (p-PLB-Ser16) (36KDa) antibody (AP0907) was purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China); SERCA2α (140KDa) antibody (9580T) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); and α-Tubulin (55 KDa) antibody (66031-1-Ig) was purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: CAR T cell enhancement by BAY 60-6583 in CD133 CAR T cells seems not to be dependent on the adenosine A2b receptor. (A, B) Blocking of the adenosine A2b receptor cannot inhibit the enhanced antitumor activities induced by BAY 60-6583. In the anti-CD133 CAR T co-culture system (E:T ratio of 4:1), the adenosine A2b receptor was blocked by 1 μM PSB603 1 h before 10 μM BAY 60-6583 or 1 μM NECA was added. 24 h later, TNF-α production (A) and tumor lysis (B) were analyzed (C) The expression of the adenosine A2b receptor on conventional (WT) and A2b receptor knockout (4 + 6) anti-CD133 CAR T cells was determined by Western blot. Data from a representative experiment of n = 4 experiments (D, E) BAY 60-6583-induced enhancement of CAR T cell-mediated antitumor activities were not influenced by adenosine A2b receptor deficiency. Conventional and A2b receptor knockout anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells at an E:T ratio of 4:1. After treatment with vehicle control or BAY 60-6583 for 24 h, cytokine secretion in the supernatant was detected (D) ; 48 h later, cytotoxicity was assessed (E) . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by 1-way (A, B) or 2-way ANOVA (D, E) . Data are represented as the mean ± SD from a representative experiment of n = 3 experiments (A, B, D, E) .

Article Snippet: After blocking with 5% milk for 1 h, the membranes were incubated with anti-adenosine A2b receptor (AAR-003, alomone labs) or anti-GAPDH (sc-32233, Santa Cruz Biotechnology) antibodies at 4°C overnight.

Techniques: Blocking Assay, Co-Culture Assay, Lysis, Expressing, Knock-Out, Western Blot, Cell Culture

Journal: Hypertension

Article Title: Hypoxia-Induced Mitogenic Factor Acts as a Nonclassical Ligand of Calcium-Sensing Receptor, Therapeutically Exploitable for Intermittent Hypoxia-Induced Pulmonary Hypertension

doi: 10.1161/hypertensionaha.116.08743

Figure Lengend Snippet: Figure 1. The immunoprecipitation screen of G protein–coupled receptor (GPCR) potentially interacted with hypoxia-induced mitogenic factor (HIMF). A, The protein lysate from pulmonary arteries (PAs) of rat after 4 weeks exposure to hypoxia was immunoprecipitated with an antibody against the GPCR of apelin receptor (APLNR), angiotensin II receptor type 1 (AGTR1), Abclonal for adrenoceptor alpha 1A (Adra1a), adenosine A2b receptor (Adora2b), calcium-sensing receptor (CaSR), endothelin receptor type A (EDNRA), 5-hydroxytryptamine receptor 2B (Htr2b), opioid receptor kappa 1 (Oprk1), purinergic receptor P2Y12 (P2ry12), prostaglandin E receptor 3 (Ptger3), or thromboxane A2 receptor (Tbxa2r), respectively, and then probed by an anti-HIMF antibody (n=3). B and C, The cross immunoprecipitation (IP) and probing with antibody against CaSR (170 KDa) and HIMF (immune blot [IB]), respectively, on lysates from pulmonary artery smooth muscle cells (PASMCs) and PAs of rats after exposure to hypoxia (n=6 for each). D and E, Immunochemical stainings on PASMCs and PAs of rats after exposure to hypoxia (n=6 and 5 for D and E, respectively).

Article Snippet: A, The protein lysate from pulmonary arteries (PAs) of rat after 4 weeks exposure to hypoxia was immunoprecipitated with an antibody against the GPCR of apelin receptor (APLNR), angiotensin II receptor type 1 (AGTR1), Abclonal for adrenoceptor alpha 1A (Adra1a), adenosine A2b receptor (Adora2b), calcium-sensing receptor (CaSR), endothelin receptor type A (EDNRA), 5-hydroxytryptamine receptor 2B (Htr2b), opioid receptor kappa 1 (Oprk1), purinergic receptor P2Y12 (P2ry12), prostaglandin E receptor 3 (Ptger3), or thromboxane A2 receptor (Tbxa2r), respectively, and then probed by an anti-HIMF antibody (n=3).

Techniques: Immunoprecipitation

Journal: Human Molecular Genetics

Article Title: Potentiation of neurotoxicity in double-mutant mice with Pink1 ablation and A53T-SNCA overexpression

doi: 10.1093/hmg/ddu520

Figure Lengend Snippet: Transcriptome validation by qPCR in DM midbrain at the age of 6 months. ( A ) The global transcriptome survey was assessed by the independent technique qPCR in midbrain of additional animals (six DM versus six WT-F1-hybrids). Tissues from an earlier age were employed to define expression dysregulations that play a role at disease stages before the formation of aggregates. This demonstrated significant downregulations for Adora2b , Dapk1 , Dcaf17 , Homer1 , Rab42 and significant upregulations for Dctn5 , Mrpl9 , Tmem181a , Xaf1 mRNA levels. The bar graphs show average 2 −ΔΔCt values ± SEM, relative to Tbp loading control, normalized to WT. These data identify factors with a role in early pathogenesis, which reflect anomalies in mitochondrial translation/apoptosis, ubiquitination, microtubular membrane transport, synaptic vesicles/receptors and DNA-damage repair. ( B ) On the basis of transcriptome bioinformatics identifying significant Lect1 transcript downregulation in the DM midbrain, qPCR was employed to demonstrate Lect1 levels to correlate inversely with the gain-of-function (left panel, five DM versus five F1-hybrid WT mice) (middle panel, four PrPmtA versus four FVB/N WT mice) and the loss-of-function (right panel, five Snca -KO versus five 129S6/SvEvTac WT mice) of alpha-synuclein in brain hemispheres of 6-month-old animals.

Article Snippet: Subsequently, expression levels were measured with the StepOnePlus Real-Time PCR System (Applied Biosystems) using cDNA from 25 ng RNA, 10 μl of FastStart Universal Probe Master (Rox) Mix (04914058001, Roche) and 1 μl of one of the following TaqMan Assays (Applied Biosystems): Adora2b (Mm00839292_m1), Dapk1 (Mm01352536_m1), Dcaf17 (Mm01334341_m1), Dctn5 (Mm00727515_s1), Homer1 (Mm00516275_m1), Mrpl9 (Mm00659648_m1), Mrpl9 (Mm00659648_m1), Pink1 (Mm00550827_m1) as independent control of genotypes, Rab42 (Mm01187370_g1), Tmem181a (Mm02581460_g1), Xaf1 (Mm01248390_m1), Lect1 (Mm00495291_m1) and Tbp (Mm00446973_m1) as endogenous control.

Techniques: Expressing

Journal: Technology in Cancer Research & Treatment

Article Title: Adenosine Receptor A2B Antagonist Inhibits the Metastasis of Gastric Cancer Cells and Enhances the Efficacy of Cisplatin

doi: 10.1177/15330338221150318

Figure Lengend Snippet: ADORA2B presents high expression in both GC tissue, greater omental metastasis tissue, and lymph node metastasis tissue than in paracancerous tissue. (A) and (C) IF analysis of ADORA2B expression in paracancerous, gastric carcinoma, and greater omental tissue. (B) and (D) IHC analyses of ADORA2B expression in paracancerous, gastric carcinoma, and greater omental metastasis and lymphatic metastasis tissue. Bar = 20μm, 400 × , ** P ＜.01, *** P ＜.001. Abbreviations: ADORA2B, adenosine A2B receptor; GC, gastric cancer; IF, immunofluorescence; IHC, immunohistochemistry.

Article Snippet: Then choose the primary antibody, ADORA2B (1:100, ER1903-44, HUABIO, China), β-catenin (1:200, ab32572, Abcam, USA), N-cadherin (1:200, ab18203, Abcam, USA), vimentin (1:100, 10366-1-AP, Proteintech, China) and E-cadherin (1:200, ab40772, Abcam, USA) incubated overnight in a 4°C humidor.

Techniques: Expressing, Immunofluorescence, Immunohistochemistry

Journal: Technology in Cancer Research & Treatment

Article Title: Adenosine Receptor A2B Antagonist Inhibits the Metastasis of Gastric Cancer Cells and Enhances the Efficacy of Cisplatin

doi: 10.1177/15330338221150318

Figure Lengend Snippet: ADORA2B Expression in Normal Tissue and Gastric Cancer Tissue.

Article Snippet: Then choose the primary antibody, ADORA2B (1:100, ER1903-44, HUABIO, China), β-catenin (1:200, ab32572, Abcam, USA), N-cadherin (1:200, ab18203, Abcam, USA), vimentin (1:100, 10366-1-AP, Proteintech, China) and E-cadherin (1:200, ab40772, Abcam, USA) incubated overnight in a 4°C humidor.

Techniques: Expressing

Journal: Technology in Cancer Research & Treatment

Article Title: Adenosine Receptor A2B Antagonist Inhibits the Metastasis of Gastric Cancer Cells and Enhances the Efficacy of Cisplatin

doi: 10.1177/15330338221150318

Figure Lengend Snippet: Relationship Between Expression of ADORA2B Protein and Clinicopathological Features of Gastric Cancer.

Article Snippet: Then choose the primary antibody, ADORA2B (1:100, ER1903-44, HUABIO, China), β-catenin (1:200, ab32572, Abcam, USA), N-cadherin (1:200, ab18203, Abcam, USA), vimentin (1:100, 10366-1-AP, Proteintech, China) and E-cadherin (1:200, ab40772, Abcam, USA) incubated overnight in a 4°C humidor.

Techniques: Expressing

Journal: Technology in Cancer Research & Treatment

Article Title: Adenosine Receptor A2B Antagonist Inhibits the Metastasis of Gastric Cancer Cells and Enhances the Efficacy of Cisplatin

doi: 10.1177/15330338221150318

Figure Lengend Snippet: The invasion and migration of GC cells were affected by ADORA2B agonists and antagonists. (A)–(D) The effects of different concentrations of an ADORA2B agonist and antagonist on the proliferation of two GC cell lines were screened by CCK8. (E)–(H) The effects on the invasion and migration of GC cells subjected to ADORA2B agonists and antagonists were examined by transwell assay; bar = 50μm, 200 × . (I)–(L) The effects on the invasion and migration of GC cells subjected to ADORA2B agonists and antagonists were examined by Wound-Healing Scratch assay. Bar = 200μm, 4 × , ** P ＜.01, *** P ＜.001. Abbreviations: ADORA2B, adenosine A2B receptor; CCK8, Cell Counting Kit-8; GC, gastric cancer.

Article Snippet: Then choose the primary antibody, ADORA2B (1:100, ER1903-44, HUABIO, China), β-catenin (1:200, ab32572, Abcam, USA), N-cadherin (1:200, ab18203, Abcam, USA), vimentin (1:100, 10366-1-AP, Proteintech, China) and E-cadherin (1:200, ab40772, Abcam, USA) incubated overnight in a 4°C humidor.

Techniques: Migration, Transwell Assay, Wound Healing Assay, Cell Counting

Journal: Technology in Cancer Research & Treatment

Article Title: Adenosine Receptor A2B Antagonist Inhibits the Metastasis of Gastric Cancer Cells and Enhances the Efficacy of Cisplatin

doi: 10.1177/15330338221150318

Figure Lengend Snippet: If analyses of ADORA2B regulated the expression of EMT marker proteins. (A and G) IF staining analysis the expression of ADORA2B, β-catenin, N-cadherin, vimentin, and E-cadherin in GC cells (AGS and HGC-27), under the regulation of ADORA2B agonists and antagonists. Quantification of the expression of (B and H) ADORA2B, (C and I) β-catenin, (D and J) vimentin, (E and K) E-cadherin, and (F and L) N-cadherin in GC cells (AGS and HGC-27) under the regulation of ADORA2B agonists and antagonists. Bar = 20μm, 400 × , ** P ＜.01, *** P ＜.001. Abbreviations: ADORA2B, adenosine A2B receptor; EMT, epithelial–mesenchymal transition; GC, gastric cancer; IF, immunofluorescence.

Article Snippet: Then choose the primary antibody, ADORA2B (1:100, ER1903-44, HUABIO, China), β-catenin (1:200, ab32572, Abcam, USA), N-cadherin (1:200, ab18203, Abcam, USA), vimentin (1:100, 10366-1-AP, Proteintech, China) and E-cadherin (1:200, ab40772, Abcam, USA) incubated overnight in a 4°C humidor.

Techniques: Expressing, Marker, Staining, Immunofluorescence

Journal: Technology in Cancer Research & Treatment

Article Title: Adenosine Receptor A2B Antagonist Inhibits the Metastasis of Gastric Cancer Cells and Enhances the Efficacy of Cisplatin

doi: 10.1177/15330338221150318

Figure Lengend Snippet: IHC analyses of ADORA2B regulated the expression of EMT marker proteins. (A and G) IHC detected the expression of ADORA2B, β-catenin, N-cadherin, vimentin, and E-cadherin in GC cells (AGS and HGC-27), under the regulation of ADORA2B agonists and antagonists. Quantification of the expression of (B and H) ADORA2B, (C and I) β-catenin, (D and J) vimentin, (E and K) E-cadherin, and (F and L) N-cadherin in GC cells (AGS and HGC-27), under the regulation of ADORA2B agonists and antagonists. Bar = 20μm, 400 × , * P ＜.05, ** P ＜.01, *** P ＜.001. Abbreviations: ADORA2B, adenosine A2B receptor; EMT, epithelial–mesenchymal transition; GC, gastric cancer; IHC, immunohistochemistry.

Article Snippet: Then choose the primary antibody, ADORA2B (1:100, ER1903-44, HUABIO, China), β-catenin (1:200, ab32572, Abcam, USA), N-cadherin (1:200, ab18203, Abcam, USA), vimentin (1:100, 10366-1-AP, Proteintech, China) and E-cadherin (1:200, ab40772, Abcam, USA) incubated overnight in a 4°C humidor.

Techniques: Expressing, Marker, Immunohistochemistry

Journal: Technology in Cancer Research & Treatment

Article Title: Adenosine Receptor A2B Antagonist Inhibits the Metastasis of Gastric Cancer Cells and Enhances the Efficacy of Cisplatin

doi: 10.1177/15330338221150318

Figure Lengend Snippet: ADORA2B agonists and antagonists regulate the expression of EMT marker proteins. (A, C, and F) Western blotting assay analyses the expression of ADORA2B, β-catenin, vimentin, and N-cadherin in AGS under treatment of ADORA2B agonists and antagonists. (B, and G–J) Western blotting assay analyses the expression of ADORA2B, β-catenin, vimentin, and N-cadherin in HGC-27 under treatment of ADORA2B agonists and antagonists. * P ＜.05, ** P ＜.01, *** P ＜.001. Abbreviations: ADORA2B, adenosine A2B receptor; EMT, epithelial–mesenchymal transition.

Article Snippet: Then choose the primary antibody, ADORA2B (1:100, ER1903-44, HUABIO, China), β-catenin (1:200, ab32572, Abcam, USA), N-cadherin (1:200, ab18203, Abcam, USA), vimentin (1:100, 10366-1-AP, Proteintech, China) and E-cadherin (1:200, ab40772, Abcam, USA) incubated overnight in a 4°C humidor.

Techniques: Expressing, Marker, Western Blot

Journal: Technology in Cancer Research & Treatment

Article Title: Adenosine Receptor A2B Antagonist Inhibits the Metastasis of Gastric Cancer Cells and Enhances the Efficacy of Cisplatin

doi: 10.1177/15330338221150318

Figure Lengend Snippet: The combined application of antagonists and cisplatin resulted in reduction in EMT marker proteins expression. (A and C–F) Western blotting assay analyses the expression of ADORA2B, β-catenin, vimentin, and N-cadherin in AGS under treatment of ADORA2B antagonists and combined application of antagonists and cisplatin. (B and G–J) Western blotting assay analyses the expression of ADORA2B, β-catenin, vimentin, and N-cadherin in HGC-27 under treatment of ADORA2B antagonists and combined application of antagonists and cisplatin. * P ＜.05, ** P ＜.01, *** P ＜.001. Abbreviations: ADORA2B, adenosine A2B receptor; EMT, epithelial–mesenchymal transition.

Article Snippet: Then choose the primary antibody, ADORA2B (1:100, ER1903-44, HUABIO, China), β-catenin (1:200, ab32572, Abcam, USA), N-cadherin (1:200, ab18203, Abcam, USA), vimentin (1:100, 10366-1-AP, Proteintech, China) and E-cadherin (1:200, ab40772, Abcam, USA) incubated overnight in a 4°C humidor.

Techniques: Marker, Expressing, Western Blot