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: 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.

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

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: 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