Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: Validation of the interaction between CtsD and the A 2A R C-terminal tail in yeast. A , transformation of Yeast 2H Gold strain with pGBKT7, pGBKT7-A 2A R 284–410 (bait), pGADT7 and pGADT7-CtsD 321–410 (prey) vectors in a pairwise combination, according to the numbered yeast line. The yeast cells were grown on SD/Leu − Trp − or SD/Leu − Trp − Ade − His − (in the presence of Aureobasidin and X-α-Gal) selective medium. pGBKT7-p53 and pGADT7-SV40 small T antigen were used as positive controls and pGBKT7-p53 and pGADT7-Lamin expressing vectors were used as negative controls. B , presentation of the different maturation forms of CtsD. Characterization of A 2A R 284–410 interactor clones that encode the different ORFs of the CtsD. The amino acid numbers of pre–pro-CtsD protein (P18242) is based on UniProtKB/Swiss-Prot databases. S: N-terminal secretion signal peptide (20 amino acids [AA]); P: propeptide (44 AA); C: cleavage site between the light and heavy chain of CtsD; arrows indicate the processing sites; D: catalytic Asp residues (D97 and D295) are depicted by black triangles ; N: N-linked glycosylation sites at Asn residues (N134 and N261) are depicted by empty triangles . A 2A R, adenosine A 2A receptor; CtsD, cathepsin D.

Article Snippet: : M5546 and I5006; Sigma–Aldrich) or 8.5 μg anti-A 2A R (catalog no.: AAR-002; Alomone Labs) were added to the complex and incubated overnight with rotation at 4 °C.

Techniques: Transformation Assay, Expressing, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: Validation of the interaction between CtsD and the A 2A R in macrophages. A , RAW 264.7 cells were transfected with a pCMV-cMyc-A 2A R 284–410 construct. The recombinant cMyc-A 2A R 284–410 and the endogenous CtsD were coimmunoprecipitated in RAW 264.7 cells. Specific bands were detected in the immune complex by WB using cMyc and CtsD antibodies. The IP was carried out using 10 μg of specific antibody in the sample (S 1 and S 2 ) and the same amount of isotype control IgG 1 (C 1 ) and goat control serum (C 2 ) in the control experiments. For loading controls ( lower panel ), 10 μg of total protein were analyzed in each lane. CtsD PF, proform; IF, intermediate form; MF-HC, mature form of heavy chain; St denotes Precision Plus Protein Dual Color Standards; and IgG HC, LC denotes immunoglobulin heavy and light chains, respectively. B , colocalization of cMyc-A 2A R 284–410 and endogenous CtsD in RAW 264.7 cells by IS. CtsD-specific and cMyc-specific primary antibodies were used to identify target proteins. The secondary antibodies were labeled with Alexa-647 ( red ) and Alexa-546 ( green ), respectively, and specific labeling was visualized by confocal microscopy (Leica TCS SP8). C , colocalization of endogenous A 2A R and CtsD was detected in mouse IPMΦ cells by IS. CtsD-specific and A 2A R-specific primary antibodies were used for the identification of target proteins. The secondary antibodies were labeled with Alexa-647 ( red ) and Alexa-546 ( green ), respectively, and the nucleus was stained with DAPI. The images were captured by confocal microscopy (Leica TCS SP8). D , endogenous CtsD was pulled down by recombinant GST-A 2A R 284–410 but not by GST alone in the cell lysate of IPMΦs. In the cell lysate, 10 μg of total protein were incubated with GST and GST-A 2A R 284–410 recombinant proteins, and the binding of CtsD was detected using CtsD-specific antibody. GST or GST-A 2A R 284–410 recombinant proteins in each lane were detected with GST-specific antibodies ( lower panel ). CtsD PF, proform; IF, intermediate form; MF-HC, mature form of heavy chain; St denotes Precision Plus Protein Dual Color Standards. E , the endogenous A 2A R and the CtsD were coimmunoprecipitated in RAW 264.7 cells. Specific bands were detected in the immune complex by WB using CtsD antibodies. The IP was carried out using 8.5 μg of A 2A R-specific antibody in the sample (S 1 –S 4 ), and antibody was not added in the control experiment ( C ). For loading controls ( right panel ), 10 μg of total protein were analyzed in each lane. CtsD PF, proform; IF, intermediate form; MF-HC, mature form of heavy chain; St denotes Precision Plus Protein Dual Color Standards; IgG HC, immunoglobulin heavy chain, respectively. Densitometry analysis data are presented as mean ± SD of three independent experiments. Values from ANOVA: F = 9.217 and p = 0.0057 for total CtsD and F = 18.59 and p = 0.0006 for CtsD HC. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus control (in the absence of A 2A R-specific antibody). A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; DAPI, 4′,6-diamidino-2-phenylindole; GST, glutathione- S -transferase; IP, immunoprecipitation; IPMΦ, mouse peritoneal macrophage; IS, immunostaining; pCMV, plasmid cytomegalovirus; WB, Western blot.

Article Snippet: : M5546 and I5006; Sigma–Aldrich) or 8.5 μg anti-A 2A R (catalog no.: AAR-002; Alomone Labs) were added to the complex and incubated overnight with rotation at 4 °C.

Techniques: Transfection, Construct, Recombinant, Labeling, Confocal Microscopy, Staining, Incubation, Binding Assay, Immunoprecipitation, Immunostaining, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: The A 2A R is a potential substrate of CtsD. A , lysates from HEK-293-FLAG-A 2A R SNAP cells were incubated in the absence and presence of recombinant mouse CtsD (1.5 μg/ml) for 1 and 2 h. The specific bands were detected by WB using FLAG-tag and β-actin-specific antibodies. Densitometry analysis data are presented as mean ± SD of three independent experiments. Values from ANOVA: F = 3.385 and p = 0.0298. ∗ p < 0.05 versus control (in the absence of recombinant mouse CtsD). B , 200 ng of recombinant GST-A 2A R 284–410 and GST proteins were incubated in the absence and presence of recombinant mouse CtsD (10 μg/ml) for 5, 10, and 30 min. The bands of the recombinant proteins were detected in polyacrylamide gel by silver staining. St denotes Precision Plus Protein Dual Color Standards. A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; GST, glutathione- S -transferase; HEK-293, human embryonic kidney 293 cell line; WB, Western blot.

Article Snippet: : M5546 and I5006; Sigma–Aldrich) or 8.5 μg anti-A 2A R (catalog no.: AAR-002; Alomone Labs) were added to the complex and incubated overnight with rotation at 4 °C.

Techniques: Incubation, Recombinant, FLAG-tag, Silver Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: Pepstatin A penetratin increases A 2A R expression in mouse macrophages. IPMΦ cells were treated with Pepstatin A penetratin (1, 3, and 9 μM) in the absence or presence of LPS (100 ng/ml) for 4 h. A , immunofluorescence detection of A 2A R in IPMΦ cells. A 2A R was stained using a specific primary antibody, and Alexa-488–conjugated secondary antibody demonstrates the localization of endogenous A 2A R protein ( green ). Nuclei of macrophages were stained with DAPI ( blue ). Images were acquired using the Opera Phenix High Content Confocal System (PerkinElmer); 190 fields and 2000 to 3000 cells were acquired per well, and laser-based autofocus was performed for each imaging position. Images of DAPI and Alexa-488 channels were collected at 2 μm of Z image plane using a 63× water immersion objective (NA, 1.15) to visualize the cells and the localization of A 2A R. B , the primary data were analyzed using Harmony 4.8 software (PerkinElmer). Spot Analyses Ready to Made Solution ( http://www.perkinelmer.com/product/harmony-4-2-office-hh17000001 ) was used with custom modifications. Image intensities were rescaled, and cells were identified using the DAPI signal. Cellular phenotypes were characterized based on the Alexa-488 signal. Cellular features, such as the number of spots, relative spot intensity in the membrane, and cytoplasm regions, were extracted. Statistical analyses of parallel datasets were made using GraphPad Prism 8 program. The evaluation of the data based on the individual analyses of 2000 to 3000 different cells is presented as mean ± SD. Values from ANOVA: F = 20.53 and p < 0.001, F = 24.78 and p < 0.001 for number of spots in membrane, cytoplasm region, respectively; F = 9.52 and p < 0.001, F = 35.28 and p < 0.001 for relative spot intensity in membrane, cytoplasm region, respectively. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus control (vehicle treated); ## p < 0.01, ### p < 0.001 versus LPS-treated cells. A 2A R, adenosine A 2A receptor; DAPI, 4′,6-diamidino-2-phenylindole; IPMΦ, mouse peritoneal macrophage; LPS, lipopolysaccharide; NA, numerical aperture.

Article Snippet: : M5546 and I5006; Sigma–Aldrich) or 8.5 μg anti-A 2A R (catalog no.: AAR-002; Alomone Labs) were added to the complex and incubated overnight with rotation at 4 °C.

Techniques: Expressing, Immunofluorescence, Staining, Imaging, Software

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: Pepstatin A penetratin, an aspartyl protease–specific inhibitor, modifies A 2A R-mediated cytokine production in LPS-activated macrophages. A , IL-1 and B , IL-6 in Pepstatin A penetratin treated IPMΦ cells after 4 h of LPS activation. Cytokine levels were determined using ELISA. Data are presented as mean ± SD of three independent experiments. Values from ANOVA: F = 66.34 and p < 0.001 for IL-6 and F = 24.44 and p < 0.001 for IL-10. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus control (vehicle treated); ## p < 0.01, ### p < 0.001 versus LPS-treated cells. A 2A R, adenosine A 2A receptor; IL, interleukin; IPMΦ, mouse peritoneal macrophage; LPS, lipopolysaccharide.

Article Snippet: : M5546 and I5006; Sigma–Aldrich) or 8.5 μg anti-A 2A R (catalog no.: AAR-002; Alomone Labs) were added to the complex and incubated overnight with rotation at 4 °C.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: A 2A R activation increases the maturation and enzyme activity of CtsD in macrophages. A , protein samples were isolated from RAW 264.7 cells after LPS activation and treatment with the A 2A R agonist CGS21680. B , protein samples were isolated from mouse IPMΦ cells after the same treatment as in A . About 10 μg of total protein sample in each lane were analyzed in duplicate by WB using CtsD-specific polyclonal antibody. Sample loading was normalized for β-actin. Statistical analyses of the relative expression of CtsD heavy chain (HC) are based on three independent experiments. Data are presented as mean ± SD. Values from ANOVA: F = 9.641 and p < 0.001 for IPMΦ and F = 22.91 and p < 0.001 for RAW 264.7 cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus LPS-activated cells. C , secreted and D , cytosolic aspartyl protease–specific activity of A 2A R agonist-treated LPS-activated IPMΦ cells. Statistical evaluation of aspartyl protease–specific activity is based on three independent experiments. Data are presented as mean ± SD. Values from ANOVA: F = 15.34 and p < 0.001 for secreted and F = 14.74 and p < 0.001 for cytosolic aspartyl protease–specific activity. ∗ p < 0.05; ∗∗∗ p < 0.001 versus control (vehicle treated); ## p < 0.01, ### p < 0.001 versus LPS-activated cells. A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; IPMΦ, mouse peritoneal macrophage; LPS, lipopolysaccharide; WB, Western blot.

Article Snippet: : M5546 and I5006; Sigma–Aldrich) or 8.5 μg anti-A 2A R (catalog no.: AAR-002; Alomone Labs) were added to the complex and incubated overnight with rotation at 4 °C.

Techniques: Activation Assay, Activity Assay, Isolation, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: A 2A R −/− mice have decreased CtsD protein expression in IPMΦ cells. Levels of different CtsD forms (PF, IF, and HC) and β-actin were measured in protein extracts from WT and A 2A R −/− mice in IPMΦ cells, using a CtsD-specific polyclonal antibody. Sample loading was normalized to β-actin. Statistical analyses of the relative expression of CtsD total proteins and HC form were based on three independent samples evaluated by independent t test. Data are presented as mean ± SD. ∗ p < 0.0145, ∗∗ p < 0.0074 versus control (A 2A R +/+ ) cells. A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; HC, heavy chain; IF, intermediate form; IPMΦ, mouse peritoneal macrophage cell; PF, proform.

Article Snippet: : M5546 and I5006; Sigma–Aldrich) or 8.5 μg anti-A 2A R (catalog no.: AAR-002; Alomone Labs) were added to the complex and incubated overnight with rotation at 4 °C.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: A 2A R activation modifies the localization of CtsD in LPS-activated macrophages. A , mouse IPMΦs were treated with CGS21680 (30 nM) in the absence or the presence of LPS (100 ng/ml) for 4 h. IS of mouse IPMΦs was conducted to determine the localization of endogenous CtsD protein ( green ) using CtsD-specific primary antibody and Alexa-488–conjugated secondary antibody. Nuclei of macrophages were stained with TO-PRO-3 iodide ( red ). Representative pictures are shown from 4 to 5 independent experiments. B , quantification of different parameters of the phagosomes from the immunostained IPMΦs was done by LAS X software (Leica Microsystem GmbH). The analysis was based on the individual densitometry of 50 different cells from 4 to 5 independent experiments. Data are presented as mean ± SD. Values from ANOVA: F = 52.44 and p < 0.001 for fluorescence intensity of and F = 9.022 and p = 0.0012 for average number of phagosomes. ∗ p < 0.05 versus control (vehicle treated); ### p < 0.001 versus LPS-activated cells. A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; IPMΦ, mouse peritoneal macrophage cell; IS, immunostaining; LPS, lipopolysaccharide.

Article Snippet: : M5546 and I5006; Sigma–Aldrich) or 8.5 μg anti-A 2A R (catalog no.: AAR-002; Alomone Labs) were added to the complex and incubated overnight with rotation at 4 °C.

Techniques: Activation Assay, Staining, Software, Fluorescence, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: Validation of the interaction between CtsD and the A 2A R C-terminal tail in yeast. A , transformation of Yeast 2H Gold strain with pGBKT7, pGBKT7-A 2A R 284–410 (bait), pGADT7 and pGADT7-CtsD 321–410 (prey) vectors in a pairwise combination, according to the numbered yeast line. The yeast cells were grown on SD/Leu − Trp − or SD/Leu − Trp − Ade − His − (in the presence of Aureobasidin and X-α-Gal) selective medium. pGBKT7-p53 and pGADT7-SV40 small T antigen were used as positive controls and pGBKT7-p53 and pGADT7-Lamin expressing vectors were used as negative controls. B , presentation of the different maturation forms of CtsD. Characterization of A 2A R 284–410 interactor clones that encode the different ORFs of the CtsD. The amino acid numbers of pre–pro-CtsD protein (P18242) is based on UniProtKB/Swiss-Prot databases. S: N-terminal secretion signal peptide (20 amino acids [AA]); P: propeptide (44 AA); C: cleavage site between the light and heavy chain of CtsD; arrows indicate the processing sites; D: catalytic Asp residues (D97 and D295) are depicted by black triangles ; N: N-linked glycosylation sites at Asn residues (N134 and N261) are depicted by empty triangles . A 2A R, adenosine A 2A receptor; CtsD, cathepsin D.

Article Snippet: The A 2A R was detected by live-cell staining using an anti-A 2A R antibody (catalog no.: AAR-002; Alomone Labs) at a concentration of 2 μg/ml.

Techniques: Transformation Assay, Expressing, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: Validation of the interaction between CtsD and the A 2A R in macrophages. A , RAW 264.7 cells were transfected with a pCMV-cMyc-A 2A R 284–410 construct. The recombinant cMyc-A 2A R 284–410 and the endogenous CtsD were coimmunoprecipitated in RAW 264.7 cells. Specific bands were detected in the immune complex by WB using cMyc and CtsD antibodies. The IP was carried out using 10 μg of specific antibody in the sample (S 1 and S 2 ) and the same amount of isotype control IgG 1 (C 1 ) and goat control serum (C 2 ) in the control experiments. For loading controls ( lower panel ), 10 μg of total protein were analyzed in each lane. CtsD PF, proform; IF, intermediate form; MF-HC, mature form of heavy chain; St denotes Precision Plus Protein Dual Color Standards; and IgG HC, LC denotes immunoglobulin heavy and light chains, respectively. B , colocalization of cMyc-A 2A R 284–410 and endogenous CtsD in RAW 264.7 cells by IS. CtsD-specific and cMyc-specific primary antibodies were used to identify target proteins. The secondary antibodies were labeled with Alexa-647 ( red ) and Alexa-546 ( green ), respectively, and specific labeling was visualized by confocal microscopy (Leica TCS SP8). C , colocalization of endogenous A 2A R and CtsD was detected in mouse IPMΦ cells by IS. CtsD-specific and A 2A R-specific primary antibodies were used for the identification of target proteins. The secondary antibodies were labeled with Alexa-647 ( red ) and Alexa-546 ( green ), respectively, and the nucleus was stained with DAPI. The images were captured by confocal microscopy (Leica TCS SP8). D , endogenous CtsD was pulled down by recombinant GST-A 2A R 284–410 but not by GST alone in the cell lysate of IPMΦs. In the cell lysate, 10 μg of total protein were incubated with GST and GST-A 2A R 284–410 recombinant proteins, and the binding of CtsD was detected using CtsD-specific antibody. GST or GST-A 2A R 284–410 recombinant proteins in each lane were detected with GST-specific antibodies ( lower panel ). CtsD PF, proform; IF, intermediate form; MF-HC, mature form of heavy chain; St denotes Precision Plus Protein Dual Color Standards. E , the endogenous A 2A R and the CtsD were coimmunoprecipitated in RAW 264.7 cells. Specific bands were detected in the immune complex by WB using CtsD antibodies. The IP was carried out using 8.5 μg of A 2A R-specific antibody in the sample (S 1 –S 4 ), and antibody was not added in the control experiment ( C ). For loading controls ( right panel ), 10 μg of total protein were analyzed in each lane. CtsD PF, proform; IF, intermediate form; MF-HC, mature form of heavy chain; St denotes Precision Plus Protein Dual Color Standards; IgG HC, immunoglobulin heavy chain, respectively. Densitometry analysis data are presented as mean ± SD of three independent experiments. Values from ANOVA: F = 9.217 and p = 0.0057 for total CtsD and F = 18.59 and p = 0.0006 for CtsD HC. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus control (in the absence of A 2A R-specific antibody). A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; DAPI, 4′,6-diamidino-2-phenylindole; GST, glutathione- S -transferase; IP, immunoprecipitation; IPMΦ, mouse peritoneal macrophage; IS, immunostaining; pCMV, plasmid cytomegalovirus; WB, Western blot.

Article Snippet: The A 2A R was detected by live-cell staining using an anti-A 2A R antibody (catalog no.: AAR-002; Alomone Labs) at a concentration of 2 μg/ml.

Techniques: Transfection, Construct, Recombinant, Labeling, Confocal Microscopy, Staining, Incubation, Binding Assay, Immunoprecipitation, Immunostaining, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: The A 2A R is a potential substrate of CtsD. A , lysates from HEK-293-FLAG-A 2A R SNAP cells were incubated in the absence and presence of recombinant mouse CtsD (1.5 μg/ml) for 1 and 2 h. The specific bands were detected by WB using FLAG-tag and β-actin-specific antibodies. Densitometry analysis data are presented as mean ± SD of three independent experiments. Values from ANOVA: F = 3.385 and p = 0.0298. ∗ p < 0.05 versus control (in the absence of recombinant mouse CtsD). B , 200 ng of recombinant GST-A 2A R 284–410 and GST proteins were incubated in the absence and presence of recombinant mouse CtsD (10 μg/ml) for 5, 10, and 30 min. The bands of the recombinant proteins were detected in polyacrylamide gel by silver staining. St denotes Precision Plus Protein Dual Color Standards. A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; GST, glutathione- S -transferase; HEK-293, human embryonic kidney 293 cell line; WB, Western blot.

Article Snippet: The A 2A R was detected by live-cell staining using an anti-A 2A R antibody (catalog no.: AAR-002; Alomone Labs) at a concentration of 2 μg/ml.

Techniques: Incubation, Recombinant, FLAG-tag, Silver Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: Pepstatin A penetratin increases A 2A R expression in mouse macrophages. IPMΦ cells were treated with Pepstatin A penetratin (1, 3, and 9 μM) in the absence or presence of LPS (100 ng/ml) for 4 h. A , immunofluorescence detection of A 2A R in IPMΦ cells. A 2A R was stained using a specific primary antibody, and Alexa-488–conjugated secondary antibody demonstrates the localization of endogenous A 2A R protein ( green ). Nuclei of macrophages were stained with DAPI ( blue ). Images were acquired using the Opera Phenix High Content Confocal System (PerkinElmer); 190 fields and 2000 to 3000 cells were acquired per well, and laser-based autofocus was performed for each imaging position. Images of DAPI and Alexa-488 channels were collected at 2 μm of Z image plane using a 63× water immersion objective (NA, 1.15) to visualize the cells and the localization of A 2A R. B , the primary data were analyzed using Harmony 4.8 software (PerkinElmer). Spot Analyses Ready to Made Solution ( http://www.perkinelmer.com/product/harmony-4-2-office-hh17000001 ) was used with custom modifications. Image intensities were rescaled, and cells were identified using the DAPI signal. Cellular phenotypes were characterized based on the Alexa-488 signal. Cellular features, such as the number of spots, relative spot intensity in the membrane, and cytoplasm regions, were extracted. Statistical analyses of parallel datasets were made using GraphPad Prism 8 program. The evaluation of the data based on the individual analyses of 2000 to 3000 different cells is presented as mean ± SD. Values from ANOVA: F = 20.53 and p < 0.001, F = 24.78 and p < 0.001 for number of spots in membrane, cytoplasm region, respectively; F = 9.52 and p < 0.001, F = 35.28 and p < 0.001 for relative spot intensity in membrane, cytoplasm region, respectively. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus control (vehicle treated); ## p < 0.01, ### p < 0.001 versus LPS-treated cells. A 2A R, adenosine A 2A receptor; DAPI, 4′,6-diamidino-2-phenylindole; IPMΦ, mouse peritoneal macrophage; LPS, lipopolysaccharide; NA, numerical aperture.

Article Snippet: The A 2A R was detected by live-cell staining using an anti-A 2A R antibody (catalog no.: AAR-002; Alomone Labs) at a concentration of 2 μg/ml.

Techniques: Expressing, Immunofluorescence, Staining, Imaging, Software

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: Pepstatin A penetratin, an aspartyl protease–specific inhibitor, modifies A 2A R-mediated cytokine production in LPS-activated macrophages. A , IL-1 and B , IL-6 in Pepstatin A penetratin treated IPMΦ cells after 4 h of LPS activation. Cytokine levels were determined using ELISA. Data are presented as mean ± SD of three independent experiments. Values from ANOVA: F = 66.34 and p < 0.001 for IL-6 and F = 24.44 and p < 0.001 for IL-10. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus control (vehicle treated); ## p < 0.01, ### p < 0.001 versus LPS-treated cells. A 2A R, adenosine A 2A receptor; IL, interleukin; IPMΦ, mouse peritoneal macrophage; LPS, lipopolysaccharide.

Article Snippet: The A 2A R was detected by live-cell staining using an anti-A 2A R antibody (catalog no.: AAR-002; Alomone Labs) at a concentration of 2 μg/ml.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: A 2A R activation increases the maturation and enzyme activity of CtsD in macrophages. A , protein samples were isolated from RAW 264.7 cells after LPS activation and treatment with the A 2A R agonist CGS21680. B , protein samples were isolated from mouse IPMΦ cells after the same treatment as in A . About 10 μg of total protein sample in each lane were analyzed in duplicate by WB using CtsD-specific polyclonal antibody. Sample loading was normalized for β-actin. Statistical analyses of the relative expression of CtsD heavy chain (HC) are based on three independent experiments. Data are presented as mean ± SD. Values from ANOVA: F = 9.641 and p < 0.001 for IPMΦ and F = 22.91 and p < 0.001 for RAW 264.7 cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus LPS-activated cells. C , secreted and D , cytosolic aspartyl protease–specific activity of A 2A R agonist-treated LPS-activated IPMΦ cells. Statistical evaluation of aspartyl protease–specific activity is based on three independent experiments. Data are presented as mean ± SD. Values from ANOVA: F = 15.34 and p < 0.001 for secreted and F = 14.74 and p < 0.001 for cytosolic aspartyl protease–specific activity. ∗ p < 0.05; ∗∗∗ p < 0.001 versus control (vehicle treated); ## p < 0.01, ### p < 0.001 versus LPS-activated cells. A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; IPMΦ, mouse peritoneal macrophage; LPS, lipopolysaccharide; WB, Western blot.

Article Snippet: The A 2A R was detected by live-cell staining using an anti-A 2A R antibody (catalog no.: AAR-002; Alomone Labs) at a concentration of 2 μg/ml.

Techniques: Activation Assay, Activity Assay, Isolation, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: A 2A R −/− mice have decreased CtsD protein expression in IPMΦ cells. Levels of different CtsD forms (PF, IF, and HC) and β-actin were measured in protein extracts from WT and A 2A R −/− mice in IPMΦ cells, using a CtsD-specific polyclonal antibody. Sample loading was normalized to β-actin. Statistical analyses of the relative expression of CtsD total proteins and HC form were based on three independent samples evaluated by independent t test. Data are presented as mean ± SD. ∗ p < 0.0145, ∗∗ p < 0.0074 versus control (A 2A R +/+ ) cells. A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; HC, heavy chain; IF, intermediate form; IPMΦ, mouse peritoneal macrophage cell; PF, proform.

Article Snippet: The A 2A R was detected by live-cell staining using an anti-A 2A R antibody (catalog no.: AAR-002; Alomone Labs) at a concentration of 2 μg/ml.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: A 2A R activation modifies the localization of CtsD in LPS-activated macrophages. A , mouse IPMΦs were treated with CGS21680 (30 nM) in the absence or the presence of LPS (100 ng/ml) for 4 h. IS of mouse IPMΦs was conducted to determine the localization of endogenous CtsD protein ( green ) using CtsD-specific primary antibody and Alexa-488–conjugated secondary antibody. Nuclei of macrophages were stained with TO-PRO-3 iodide ( red ). Representative pictures are shown from 4 to 5 independent experiments. B , quantification of different parameters of the phagosomes from the immunostained IPMΦs was done by LAS X software (Leica Microsystem GmbH). The analysis was based on the individual densitometry of 50 different cells from 4 to 5 independent experiments. Data are presented as mean ± SD. Values from ANOVA: F = 52.44 and p < 0.001 for fluorescence intensity of and F = 9.022 and p = 0.0012 for average number of phagosomes. ∗ p < 0.05 versus control (vehicle treated); ### p < 0.001 versus LPS-activated cells. A 2A R, adenosine A 2A receptor; CtsD, cathepsin D; IPMΦ, mouse peritoneal macrophage cell; IS, immunostaining; LPS, lipopolysaccharide.

Article Snippet: The A 2A R was detected by live-cell staining using an anti-A 2A R antibody (catalog no.: AAR-002; Alomone Labs) at a concentration of 2 μg/ml.

Techniques: Activation Assay, Staining, Software, Fluorescence, Immunostaining

Journal: Purinergic Signalling

Article Title: Adenosine receptor expression in the adult zebrafish retina

doi: 10.1007/s11302-019-09667-0

Figure Lengend Snippet: Description of primer sequences and cDNA sources for the adora genes in zebrafish

Article Snippet: Antibodies Primary polyclonal antibodies targeting adenosine receptors for immunohistochemistry and western blotting experiments were purchased from Alomone Labs (Jerusalem, Israel; Adenosine Receptor Antibody Explorer Kit, cat. no. AK-105) and were raised against rabbit anti-A1R (1:200; cat. no. AAR-006; corresponds to amino acid residues 213–229 of human A1AR [accession {"type":"entrez-protein","attrs":{"text":"P30542","term_id":"231473","term_text":"P30542"}} P30542 ], epitope is located on the 3rd intracellular loop), anti-A2AR (1:200; cat. no. AAR-002; corresponds to amino acid residues 201–215 of mouse adenosine A2A receptor [accession {"type":"entrez-protein","attrs":{"text":"Q60613","term_id":"341940595","term_text":"Q60613"}} Q60613 ], epitope is located on the 3rd intracellular loop), anti-A2BR (1:200; cat. no. AAR-003; corresponds to amino acid residues 147–166 of human A2BAR with replacement of cysteine 154 with serine [accession {"type":"entrez-protein","attrs":{"text":"P29275","term_id":"112938","term_text":"P29275"}} P29275 ], epitope is located on the 2nd extracellular loop), and anti-A3R (1:1000; cat. no. AAR-004; corresponds to amino acid residues 216–230 of human A3AR [accession {"type":"entrez-protein","attrs":{"text":"P0DMS8","term_id":"803374855","term_text":"P0DMS8"}} P0DMS8 ], the epitope is located on the 3rd intracellular loop) receptor subtypes.

Techniques: Sequencing, Amplification

Journal: Purinergic Signalling

Article Title: Adenosine receptor expression in the adult zebrafish retina

doi: 10.1007/s11302-019-09667-0

Figure Lengend Snippet: Adenosine receptor expression in the zebrafish retina. In order from top to bottom, A1, A2A, A2B, and A3 receptor subtype. a mRNA was detected using in situ hybridization in retinal sections, A1 (adora1a), A2A (adora2aa), A2B (adora2b), and A3 (ENSDARG00000044061 A3 3rd homolog); parentheses denote the gene shown in the figure (see Table ​Table1).1). Specific adenosine receptor antibody signal (white arrows) was determined in the presence and absence of the corresponding peptide in b retinal cryosections using immunohistochemistry, where blue nuclei have been labeled with RedDot1, and c retinal homogenate using western blotting. RPE retinal pigment epithelium, OS outer segment, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer, PB peptide block, MW molecular weight. For in situ, n = 6 retinas, N = 3 zebrafish. For immunohistochemistry, n = 14 retinas, N = 7 zebrafish. For western blotting, n = 10 retinas, N = 5 zebrafish. Scale = 20 μm

Article Snippet: Antibodies Primary polyclonal antibodies targeting adenosine receptors for immunohistochemistry and western blotting experiments were purchased from Alomone Labs (Jerusalem, Israel; Adenosine Receptor Antibody Explorer Kit, cat. no. AK-105) and were raised against rabbit anti-A1R (1:200; cat. no. AAR-006; corresponds to amino acid residues 213–229 of human A1AR [accession {"type":"entrez-protein","attrs":{"text":"P30542","term_id":"231473","term_text":"P30542"}} P30542 ], epitope is located on the 3rd intracellular loop), anti-A2AR (1:200; cat. no. AAR-002; corresponds to amino acid residues 201–215 of mouse adenosine A2A receptor [accession {"type":"entrez-protein","attrs":{"text":"Q60613","term_id":"341940595","term_text":"Q60613"}} Q60613 ], epitope is located on the 3rd intracellular loop), anti-A2BR (1:200; cat. no. AAR-003; corresponds to amino acid residues 147–166 of human A2BAR with replacement of cysteine 154 with serine [accession {"type":"entrez-protein","attrs":{"text":"P29275","term_id":"112938","term_text":"P29275"}} P29275 ], epitope is located on the 2nd extracellular loop), and anti-A3R (1:1000; cat. no. AAR-004; corresponds to amino acid residues 216–230 of human A3AR [accession {"type":"entrez-protein","attrs":{"text":"P0DMS8","term_id":"803374855","term_text":"P0DMS8"}} P0DMS8 ], the epitope is located on the 3rd intracellular loop) receptor subtypes.

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Labeling, Western Blot, Blocking Assay, Molecular Weight, In Situ

Journal: Purinergic Signalling

Article Title: Adenosine receptor expression in the adult zebrafish retina

doi: 10.1007/s11302-019-09667-0

Figure Lengend Snippet: A1 and A2A receptors are expressed on ON cone bipolar cell terminals. a A1 and b A2A receptors are expressed around ON cone bipolar cell terminals (white arrowheads), but little to no expression is present in the mixed rod/cone response bipolar cells (Mb-1; yellow arrowheads), where blue nuclei have been labeled with RedDot1. OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer; n = 12 retinas, N = 6 zebrafish. Scale = 5 μm

Article Snippet: Antibodies Primary polyclonal antibodies targeting adenosine receptors for immunohistochemistry and western blotting experiments were purchased from Alomone Labs (Jerusalem, Israel; Adenosine Receptor Antibody Explorer Kit, cat. no. AK-105) and were raised against rabbit anti-A1R (1:200; cat. no. AAR-006; corresponds to amino acid residues 213–229 of human A1AR [accession {"type":"entrez-protein","attrs":{"text":"P30542","term_id":"231473","term_text":"P30542"}} P30542 ], epitope is located on the 3rd intracellular loop), anti-A2AR (1:200; cat. no. AAR-002; corresponds to amino acid residues 201–215 of mouse adenosine A2A receptor [accession {"type":"entrez-protein","attrs":{"text":"Q60613","term_id":"341940595","term_text":"Q60613"}} Q60613 ], epitope is located on the 3rd intracellular loop), anti-A2BR (1:200; cat. no. AAR-003; corresponds to amino acid residues 147–166 of human A2BAR with replacement of cysteine 154 with serine [accession {"type":"entrez-protein","attrs":{"text":"P29275","term_id":"112938","term_text":"P29275"}} P29275 ], epitope is located on the 2nd extracellular loop), and anti-A3R (1:1000; cat. no. AAR-004; corresponds to amino acid residues 216–230 of human A3AR [accession {"type":"entrez-protein","attrs":{"text":"P0DMS8","term_id":"803374855","term_text":"P0DMS8"}} P0DMS8 ], the epitope is located on the 3rd intracellular loop) receptor subtypes.

Techniques: Expressing, Labeling

Journal: Purinergic Signalling

Article Title: Adenosine receptor expression in the adult zebrafish retina

doi: 10.1007/s11302-019-09667-0

Figure Lengend Snippet: A2A receptors are present on photoreceptors in the outer retina. A2A labeling presents as bright puncta that is localized to the outer retina and appears to be most prominent around the cone ellipsoids. There is a small amount of dull, more homogenous nonspecific labeling that is clearly distinguishable from the specific puncta labeling in the outer retina, where all puncta are absent in the presence of the peptide block. OS outer segment, IS inner segment, olm outer limiting membrane, ONL outer nuclear layer, OPL outer plexiform layer; n = 12 retinas, N = 6 zebrafish. Scale bar = 2.5 μm

Article Snippet: Antibodies Primary polyclonal antibodies targeting adenosine receptors for immunohistochemistry and western blotting experiments were purchased from Alomone Labs (Jerusalem, Israel; Adenosine Receptor Antibody Explorer Kit, cat. no. AK-105) and were raised against rabbit anti-A1R (1:200; cat. no. AAR-006; corresponds to amino acid residues 213–229 of human A1AR [accession {"type":"entrez-protein","attrs":{"text":"P30542","term_id":"231473","term_text":"P30542"}} P30542 ], epitope is located on the 3rd intracellular loop), anti-A2AR (1:200; cat. no. AAR-002; corresponds to amino acid residues 201–215 of mouse adenosine A2A receptor [accession {"type":"entrez-protein","attrs":{"text":"Q60613","term_id":"341940595","term_text":"Q60613"}} Q60613 ], epitope is located on the 3rd intracellular loop), anti-A2BR (1:200; cat. no. AAR-003; corresponds to amino acid residues 147–166 of human A2BAR with replacement of cysteine 154 with serine [accession {"type":"entrez-protein","attrs":{"text":"P29275","term_id":"112938","term_text":"P29275"}} P29275 ], epitope is located on the 2nd extracellular loop), and anti-A3R (1:1000; cat. no. AAR-004; corresponds to amino acid residues 216–230 of human A3AR [accession {"type":"entrez-protein","attrs":{"text":"P0DMS8","term_id":"803374855","term_text":"P0DMS8"}} P0DMS8 ], the epitope is located on the 3rd intracellular loop) receptor subtypes.

Techniques: Labeling, Blocking Assay