mouse cd73 gene  (Sino Biological)

Journal: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Expression and purification of CD73 protein. ( A ) E. coli Origami cells were transformed with pET28a-CD73 plasmid and plated on LB agar containing kanamycin. Six colonies were randomly picked and PCR performed with primers for CD73. Size of the product is 536 bp. L represents 100 bp plus ladder. ( B ) Transformation was further confirmed in two of the positive colonies through restriction enzyme digestion with EcoRI and XhoI, either singly (SD) or together (DD). Size of the insert (i) is 1731 bp and of vector is 5369 bp. U, undigested plasmid; M, 1 kb ladder. ( C ) SDS-PAGE analysis of the expression of rCD73 was performed from cells treated with different concentrations of IPTG and/or temperature and cultured in a shaker incubator at 300 rpm displayed nearly same level of expression in each case within the pellet. M, Molecular weight marker; S, lysate supernatant; P, lysate pellet. ( D ) The expressed protein was mostly found in the inclusion body (IB) which was washed several times to get a pure IB. ( E ) The protein in the inclusion body was solubilized in 8 M urea. ( F ) Representative Coomassie-stained SDS-PAGE gel shows the various fractions followed by the protein present in the elute in lane E. ( G ) rCD73 was purified using a Ni-NTA column in the AKTA Pure System which showed a single band. ( H ) The denatured rCD73 was refolded in a refolding buffer, concentrated using Amicon Ultracentrifuge filter with a 30-kDa cut-off, and then dialyzed to remove urea. Representative Coomassie-stained SDS-PAGE gel depicts a strong band around 63 kDa. FT, flow through; DF, dialyzed fraction. ( I ) The identity of this band was further confirmed using Western blot tested with antibody against anti-CD73. Lysate from mouse brain was used as a positive control, PC. RP, recombinant protein. ( J ) Absorbance spectra of the refolded protein is depicted in comparison to the denatured protein present in the inclusion body. A shift in the absorbance at 365 nm wavelength (see arrow) indicates protein folding. ( K ) The specific activity of the refolded recombinant protein was tested through phosphatase assay. *** p < 0.001, n = 3.

Article Snippet: The open reading frame of the mouse CD73 gene ( NM_011851.4 , NT5E gene) was commercially obtained as a pMD18-T Simple vector from SinoBiologicals (pMD-mNT5E, Cat. No. MG50231-M, Krishgen Biosystems, Mumbai, India).

Techniques: Expressing, Purification, Transformation Assay, Plasmid Preparation, SDS Page, Cell Culture, Molecular Weight, Marker, Staining, Western Blot, Positive Control, Recombinant, Comparison, Activity Assay, Phosphatase Assay

Journal: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Exogenous addition of recombinant CD73 reduces the effect of ATP on LPS-mediated COX-2 expression. ( A , B ) RAW264.7 macrophage cells were treated with ATP (100 µM), ATPγS (100 µM) or NECA (100 µM), alone or in the presence of LPS (10 ng/ml). Cells were harvested after 24 h and COX-2 expression was checked. β-actin was used as a housekeeping control. ( C ) RAW264.7 cells were treated with different doses of LPS (0.01–10 µg/ml) for 24 h. Culture media was then collected to estimate amount of ATP released. * p < 0.05 with respect to untreated cells ( n = 4). ( D , E ) J774A.1 macrophage cells were treated with LPS (10 ng/ml), alone or together with ATP (100 µM), AMP (100 µM) or NECA (100 µM). 24 h later cells were harvested and checked for COX-2 expression. Representative Western blot image is depicted ( D ) and the relative fold change, calculated by normalization of COX-2 expression with that of β-actin, from n = 3 experiments, is depicted in ( E ). * p < 0.05 with respect to control. $ p < 0.05 with respect to LPS-treated cells. ( F , G ) J774A.1 cells treated with LPS, AMP and NECA, alone or in combination were harvested after 4 h for the isolation of mRNA followed by cDNA synthesis. Real time PCR was performed to check the expression of various M1 ( F ) and M2 ( G ) markers ( n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 with respect to untreated cells. ( H ) mRNA was isolated from untreated J774A.1 cells and mouse brain tissue and cDNA synthesis was performed. Real time PCR was performed to check the expression of CD73 and CD39 using specific primers. δC t calculated by normalizing with β-actin ( n = 3). ( I , J ) J774A.1 cells were exogenously treated with rCD73 (300 ng) in the presence of ATP alone or ATP together with LPS. 24 h later cell lysates were collected for checking COX-2 expression. Representative blot shown in (I) while relative fold change calculated from normalized COX-2 expression from at least 7 experiments is given in ( J ). ** p < 0.01, *** p < 0.001 ( n = 7). ( K , L ) Cells were treated with LPS, AMP, or AMP-CP (1 µM), in the presence or absence of exogenously added rCD73 protein. After 24 h, cell lysates were used for checking the expression of COX-2. Representative blot shown in ( K ) with relative fold change from normalized COX-2 ( n = 6) given in ( L ). * p < 0.05, ** p < 0.01, *** p < 0.001 relative to the columns shown in the figure. $ p < 0.05 relative to the same stimulation but without rCD73.

Article Snippet: The open reading frame of the mouse CD73 gene ( NM_011851.4 , NT5E gene) was commercially obtained as a pMD18-T Simple vector from SinoBiologicals (pMD-mNT5E, Cat. No. MG50231-M, Krishgen Biosystems, Mumbai, India).

Techniques: Recombinant, Expressing, Control, Western Blot, Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Overexpression of CD73 in J774A.1 cells reduces COX-2 expression. ( A ) Presence of the insert (CD73 gene) in the pcDNA3.1 vector was confirmed through PCR ( A ), and restriction analysis using EcoRI and XhoI digestion ( B ). L, 1 kb plus ladder; U, undigested plasmid; SD, EcoRI-treated plasmid; DD, double digestion. Size of the insert, 1731 bp; size of vector, 5446 bp. ( C ) Following transfection, expression of CD73 checked by real time PCR. δC t calculated by normalizing with β-actin. ** p < 0.01 with respect to non-transfected cells ( n = 3). ( D , E ) CD73 expression in transfected cells was analyzed through Western blot, and relative densitometry shown after normalization with β-actin ( n = 4). ( F ) Activity of CD73 in cell lysates was checked by measuring the amount of phosphate released using AMP as a substrate. *** p < 0.001 ( n = 4). ( G ) CD73 activity was also measured in transfected cells ( n = 4). J774A.1 cells were transfected with CD73 for 28 h followed by replacing the media with a phosphate-free DMEM. AMP (100 µM), with or without AMP-CP (1 µM), was then added to the media which was collected after 1 h for the analysis of free phosphate. ( H , I ) J774A.1 cells were transfected with an empty vector (pcDNA3.1) or with CD73 for 28 h followed by stimulation with LPS (10 ng/ml), AMP (100 µM), or AMP-CP (1 µM), as indicated, for 24 h, for analysis of COX-2 protein using Western blot ( H ). Densitometric analysis ( n = 6) represents relative change in COX-2 expression as fold change with respect to empty vector-transfected control cells ( I ). * p < 0.05, ** p < 0.01, *** p < 0.001 with respected to indicated conditions. $$ p < 0.01 with respect to empty vector-transfected cells also treated with LPS and AMP. (J, K) CD73-transfected cells were stimulated with both LPS (10 ng/ml) and ATP (100 µM) for 24 h followed by immunocytochemistry using anti-COX-2 antibody (green). Cells were counterstained with the nuclear dye, Hoechst 33,342 (blue). Scale bar, 20 μm. Integrated density of COX-2 positive signal was normalized with that of Hoechst 33,342 signal and represented as % in (K). *** p < 0.001 ( n = 2).

Article Snippet: The open reading frame of the mouse CD73 gene ( NM_011851.4 , NT5E gene) was commercially obtained as a pMD18-T Simple vector from SinoBiologicals (pMD-mNT5E, Cat. No. MG50231-M, Krishgen Biosystems, Mumbai, India).

Techniques: Over Expression, Expressing, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Control, Immunocytochemistry