anti a 2a r  (Alomone Labs)


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    Structured Review

    Alomone Labs anti a 2a r
    Validation of the interaction between CtsD and the A <t>2A</t> <t>R</t> 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.
    Anti A 2a R, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling"

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.101888

    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.
    Figure Legend 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.

    Techniques Used: Transformation Assay, Expressing, Clone Assay

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Immunofluorescence, Staining, Imaging, Software

    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.
    Figure Legend 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.

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Expressing

    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.
    Figure Legend 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.

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

    anti a 2a r  (Alomone Labs)


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    Structured Review

    Alomone Labs anti a 2a r
    Validation of the interaction between CtsD and the A <t>2A</t> <t>R</t> 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.
    Anti A 2a R, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti a 2a r/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti a 2a r - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling"

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.101888

    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.
    Figure Legend 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.

    Techniques Used: Transformation Assay, Expressing, Clone Assay

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Immunofluorescence, Staining, Imaging, Software

    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.
    Figure Legend 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.

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Expressing

    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.
    Figure Legend 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.

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

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    Alomone Labs anti a 2a r antibody
    Validation of the interaction between CtsD and the A <t>2A</t> <t>R</t> 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.
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    1) Product Images from "Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling"

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.101888

    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.
    Figure Legend 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.

    Techniques Used: Transformation Assay, Expressing, Clone Assay

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Immunofluorescence, Staining, Imaging, Software

    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.
    Figure Legend 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.

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Expressing

    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.
    Figure Legend 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.

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

    anti human anti adenosine a 2a r rabbit polyclonal antibody  (Alomone Labs)


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    Alomone Labs anti human anti adenosine a 2a r rabbit polyclonal antibody
    ADORA2A siRNAs decrease ADORA2A mRNA and A <t>2A</t> <t>R</t> protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.
    Anti Human Anti Adenosine A 2a R Rabbit Polyclonal Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Targeted knockdown of the adenosine A 2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells"

    Article Title: Targeted knockdown of the adenosine A 2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2021.03.001

    ADORA2A siRNAs decrease ADORA2A mRNA and A 2A R protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.
    Figure Legend Snippet: ADORA2A siRNAs decrease ADORA2A mRNA and A 2A R protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.

    Techniques Used: Expressing, Labeling, Transfection, Standard Deviation, Flow Cytometry, Fluorescence, Staining

    CD45RO(A 2A R)-NPs rescue the chemotactic ability of HNSCC CD8 + memory T cells in the presence of adenosine (A and B) Representative two point trajectories of T cells migrating along the CXCL10 gradient (green) or CXCL10 + adenosine (ADO) gradient (blue) in HNSCC CD8 + memory T cells treated with (A) CD45RO(scr)-NPs or (B) CD45RO(A 2A R)-NPs. Trajectories artificially set to start at the origin with the red triangle representing the Y-COM. (C) Y-COM of HNSCC CD8 + memory T cells treated with (left) CD45RO(scr)-NPs (n = 6) or (right) CD45RO(A 2A R)-NPs (n = 6) in the presence of CXCL10 or CXCL10 + adenosine. Significance was determined using (left) a Wilcoxon signed rank test or (right) paired Student’s t test. (D) Percent inhibition of Y-COM of HNSCC CD8 + memory T cells in the presence of CXCL10 + adenosine compared to CXCL10 alone when treated with CD45RO(scr)-NPs (n = 6) compared to CD45RO(A 2A R)-NPs (n = 6). Data are represented single dot plots (colored dots; wherein each dot represents a single donor) and as mean ± standard deviation (shown in black). Significance was determined using Student’s t test.
    Figure Legend Snippet: CD45RO(A 2A R)-NPs rescue the chemotactic ability of HNSCC CD8 + memory T cells in the presence of adenosine (A and B) Representative two point trajectories of T cells migrating along the CXCL10 gradient (green) or CXCL10 + adenosine (ADO) gradient (blue) in HNSCC CD8 + memory T cells treated with (A) CD45RO(scr)-NPs or (B) CD45RO(A 2A R)-NPs. Trajectories artificially set to start at the origin with the red triangle representing the Y-COM. (C) Y-COM of HNSCC CD8 + memory T cells treated with (left) CD45RO(scr)-NPs (n = 6) or (right) CD45RO(A 2A R)-NPs (n = 6) in the presence of CXCL10 or CXCL10 + adenosine. Significance was determined using (left) a Wilcoxon signed rank test or (right) paired Student’s t test. (D) Percent inhibition of Y-COM of HNSCC CD8 + memory T cells in the presence of CXCL10 + adenosine compared to CXCL10 alone when treated with CD45RO(scr)-NPs (n = 6) compared to CD45RO(A 2A R)-NPs (n = 6). Data are represented single dot plots (colored dots; wherein each dot represents a single donor) and as mean ± standard deviation (shown in black). Significance was determined using Student’s t test.

    Techniques Used: Inhibition, Standard Deviation

    anti human anti adenosine a 2a r rabbit polyclonal antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti human anti adenosine a 2a r rabbit polyclonal antibody
    ADORA2A siRNAs decrease ADORA2A mRNA and A <t>2A</t> <t>R</t> protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.
    Anti Human Anti Adenosine A 2a R Rabbit Polyclonal Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human anti adenosine a 2a r rabbit polyclonal antibody/product/Alomone Labs
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    Images

    1) Product Images from "Targeted knockdown of the adenosine A 2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells"

    Article Title: Targeted knockdown of the adenosine A 2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2021.03.001

    ADORA2A siRNAs decrease ADORA2A mRNA and A 2A R protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.
    Figure Legend Snippet: ADORA2A siRNAs decrease ADORA2A mRNA and A 2A R protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.

    Techniques Used: Expressing, Labeling, Transfection, Standard Deviation, Flow Cytometry, Fluorescence, Staining

    CD45RO(A 2A R)-NPs rescue the chemotactic ability of HNSCC CD8 + memory T cells in the presence of adenosine (A and B) Representative two point trajectories of T cells migrating along the CXCL10 gradient (green) or CXCL10 + adenosine (ADO) gradient (blue) in HNSCC CD8 + memory T cells treated with (A) CD45RO(scr)-NPs or (B) CD45RO(A 2A R)-NPs. Trajectories artificially set to start at the origin with the red triangle representing the Y-COM. (C) Y-COM of HNSCC CD8 + memory T cells treated with (left) CD45RO(scr)-NPs (n = 6) or (right) CD45RO(A 2A R)-NPs (n = 6) in the presence of CXCL10 or CXCL10 + adenosine. Significance was determined using (left) a Wilcoxon signed rank test or (right) paired Student’s t test. (D) Percent inhibition of Y-COM of HNSCC CD8 + memory T cells in the presence of CXCL10 + adenosine compared to CXCL10 alone when treated with CD45RO(scr)-NPs (n = 6) compared to CD45RO(A 2A R)-NPs (n = 6). Data are represented single dot plots (colored dots; wherein each dot represents a single donor) and as mean ± standard deviation (shown in black). Significance was determined using Student’s t test.
    Figure Legend Snippet: CD45RO(A 2A R)-NPs rescue the chemotactic ability of HNSCC CD8 + memory T cells in the presence of adenosine (A and B) Representative two point trajectories of T cells migrating along the CXCL10 gradient (green) or CXCL10 + adenosine (ADO) gradient (blue) in HNSCC CD8 + memory T cells treated with (A) CD45RO(scr)-NPs or (B) CD45RO(A 2A R)-NPs. Trajectories artificially set to start at the origin with the red triangle representing the Y-COM. (C) Y-COM of HNSCC CD8 + memory T cells treated with (left) CD45RO(scr)-NPs (n = 6) or (right) CD45RO(A 2A R)-NPs (n = 6) in the presence of CXCL10 or CXCL10 + adenosine. Significance was determined using (left) a Wilcoxon signed rank test or (right) paired Student’s t test. (D) Percent inhibition of Y-COM of HNSCC CD8 + memory T cells in the presence of CXCL10 + adenosine compared to CXCL10 alone when treated with CD45RO(scr)-NPs (n = 6) compared to CD45RO(A 2A R)-NPs (n = 6). Data are represented single dot plots (colored dots; wherein each dot represents a single donor) and as mean ± standard deviation (shown in black). Significance was determined using Student’s t test.

    Techniques Used: Inhibition, Standard Deviation

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    Alomone Labs anti a 2a r
    Validation of the interaction between CtsD and the A <t>2A</t> <t>R</t> 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.
    Anti A 2a R, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti a 2a r/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    94
    Alomone Labs anti a 2a r antibody
    Validation of the interaction between CtsD and the A <t>2A</t> <t>R</t> 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.
    Anti A 2a R Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti a 2a r antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti a 2a r antibody - by Bioz Stars, 2023-01
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    93
    Alomone Labs anti human anti adenosine a 2a r rabbit polyclonal antibody
    ADORA2A siRNAs decrease ADORA2A mRNA and A <t>2A</t> <t>R</t> protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.
    Anti Human Anti Adenosine A 2a R Rabbit Polyclonal Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human anti adenosine a 2a r rabbit polyclonal antibody/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
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    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    ADORA2A siRNAs decrease ADORA2A mRNA and A 2A R protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Targeted knockdown of the adenosine A 2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells

    doi: 10.1016/j.omtm.2021.03.001

    Figure Lengend Snippet: ADORA2A siRNAs decrease ADORA2A mRNA and A 2A R protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3 + or CD8 + T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A 2A R protein expression in activated HD CD3 + or CD8 + T cells 24 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), 48 h (n = 5; 3 CD3 + T cell samples, 2 CD8 + T cell samples), or 72 h (n = 4; 2 CD3 + T cell samples, 2 CD8 + T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A 2A R expression showing the highest degree of A 2A R protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A 2A R expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A 2A R expression. The unstained sample is indicated in orange in the histogram while the A 2A R expression of cells treated with scr-RNAs is indicated in blue and the A 2A R expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A 2A R antibody specificity where CD3 + T cells were stained with A 2A R antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A 2A R antibody + peptide is indicated in red and A 2A R antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.

    Article Snippet: Anti-human anti-adenosine A 2A R rabbit polyclonal antibody (0.75 μg) was incubated with 1.5 μg A 2A R blocking peptide (Alomone) at a 1:2 ratio (1.5 μg peptide was added to 0.75 μg antibody).

    Techniques: Expressing, Labeling, Transfection, Standard Deviation, Flow Cytometry, Fluorescence, Staining

    CD45RO(A 2A R)-NPs rescue the chemotactic ability of HNSCC CD8 + memory T cells in the presence of adenosine (A and B) Representative two point trajectories of T cells migrating along the CXCL10 gradient (green) or CXCL10 + adenosine (ADO) gradient (blue) in HNSCC CD8 + memory T cells treated with (A) CD45RO(scr)-NPs or (B) CD45RO(A 2A R)-NPs. Trajectories artificially set to start at the origin with the red triangle representing the Y-COM. (C) Y-COM of HNSCC CD8 + memory T cells treated with (left) CD45RO(scr)-NPs (n = 6) or (right) CD45RO(A 2A R)-NPs (n = 6) in the presence of CXCL10 or CXCL10 + adenosine. Significance was determined using (left) a Wilcoxon signed rank test or (right) paired Student’s t test. (D) Percent inhibition of Y-COM of HNSCC CD8 + memory T cells in the presence of CXCL10 + adenosine compared to CXCL10 alone when treated with CD45RO(scr)-NPs (n = 6) compared to CD45RO(A 2A R)-NPs (n = 6). Data are represented single dot plots (colored dots; wherein each dot represents a single donor) and as mean ± standard deviation (shown in black). Significance was determined using Student’s t test.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Targeted knockdown of the adenosine A 2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells

    doi: 10.1016/j.omtm.2021.03.001

    Figure Lengend Snippet: CD45RO(A 2A R)-NPs rescue the chemotactic ability of HNSCC CD8 + memory T cells in the presence of adenosine (A and B) Representative two point trajectories of T cells migrating along the CXCL10 gradient (green) or CXCL10 + adenosine (ADO) gradient (blue) in HNSCC CD8 + memory T cells treated with (A) CD45RO(scr)-NPs or (B) CD45RO(A 2A R)-NPs. Trajectories artificially set to start at the origin with the red triangle representing the Y-COM. (C) Y-COM of HNSCC CD8 + memory T cells treated with (left) CD45RO(scr)-NPs (n = 6) or (right) CD45RO(A 2A R)-NPs (n = 6) in the presence of CXCL10 or CXCL10 + adenosine. Significance was determined using (left) a Wilcoxon signed rank test or (right) paired Student’s t test. (D) Percent inhibition of Y-COM of HNSCC CD8 + memory T cells in the presence of CXCL10 + adenosine compared to CXCL10 alone when treated with CD45RO(scr)-NPs (n = 6) compared to CD45RO(A 2A R)-NPs (n = 6). Data are represented single dot plots (colored dots; wherein each dot represents a single donor) and as mean ± standard deviation (shown in black). Significance was determined using Student’s t test.

    Article Snippet: Anti-human anti-adenosine A 2A R rabbit polyclonal antibody (0.75 μg) was incubated with 1.5 μg A 2A R blocking peptide (Alomone) at a 1:2 ratio (1.5 μg peptide was added to 0.75 μg antibody).

    Techniques: Inhibition, Standard Deviation