Journal: Scientific Reports

Article Title: Role of P2X 4 Receptor in Mouse Voiding Function

doi: 10.1038/s41598-018-20216-4

Figure Lengend Snippet: Immunoprecipitation with anti-P2X 1 antibody. Antibodies to P2X 1 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads were resolved by SDS-PAGE, and Western blots were probed with A) P2X 1, B) P2X 4 or C) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. Monomeric P2X 1 can be seen highly concentrated in the pulldown fraction at 50 kDa. Little P2X 1 appears in FT. ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type, but is completely absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to possibly P2X 1 . Note, there is no evidence of the 70 kDa band in the IP lane. ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 1 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

Article Snippet: The isolated proteins were resolved on 8–16% polyacrylamide gradient gels under reducing condition (+0.1 M DTT and 95 °C for 5 min before loading), and further blotted and probed with anti-P2X 1 , anti-P2X 4 and anti-5′-nucleotidase (NT5E) (Catalogue #: MAB44881, R&D system) antibodies for protein detection.

Techniques: Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Protein Binding

Journal: Scientific Reports

Article Title: Role of P2X 4 Receptor in Mouse Voiding Function

doi: 10.1038/s41598-018-20216-4

Figure Lengend Snippet: Immunoprecipitation with anti-P2X 4 antibody. Antibodies to P2X 4 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads, were resolved by SDS-PAGE, and Western blots were probed with A ) P2X 1, B) P2X 4 or C ) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. P2X 1 is highly concentrated in the FT fractions. Minor potential P2X 1 staining appears in the IP lane, however this is due to P2X 4 antibody cross-reacting and pulling down some P2X 1 . ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type IP lane, but is absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to P2X 1 (50 kDa band). ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 4 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

Article Snippet: The isolated proteins were resolved on 8–16% polyacrylamide gradient gels under reducing condition (+0.1 M DTT and 95 °C for 5 min before loading), and further blotted and probed with anti-P2X 1 , anti-P2X 4 and anti-5′-nucleotidase (NT5E) (Catalogue #: MAB44881, R&D system) antibodies for protein detection.

Techniques: Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Staining, Protein Binding

Journal: Cell Death & Disease

Article Title: P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: Role of the A2A/CD39/CD73 axis

doi: 10.1038/s41419-025-07897-2

Figure Lengend Snippet: A CT26 CRC cells were stained with PKH26GL (red) and quinacrine (green) fluorescent dyes. Images were acquired using confocal microscopy at time 0 and after 5 min following P2X7 activation with 300 µM BzATP and are extrapolated from a 30-min time course (see supplementary videos and ). B CT26 cells were pre-treated with P2X7 antagonist AZ10606120 (5 µM) for 10 min before application of BzATP. C Number of vesicles released in 30 min from CT26 cells in PBS vehicle (PBS S-VS), following stimulation with P2X7 agonist ATP (P2X7-VS) or 10 min pretreatment with P2X7 antagonist AZ10606120 followed by stimulation with 3 mM ATP (AZ-VS) ( n = 5). D Size of PBS S-VS, P2X7-VS, and AZ-VS ( n = 5). E Number of vesicles released in 30 min from CT26 cells in DMSO vehicle (DMSO S-VS), following stimulation with P2X7 agonist ATP (P2X7-VS) or 10 min pretreatment with P2X7 antagonist A740003 followed by stimulation with 3 mM ATP (A74-VS) ( n = 7). F Size of DMSO- S-VS, P2X7-VS, and A74-VS ( n = 7). G Western blot for GM130, Alix, P2X7, CD39, CD73, and A2A in CT26 cells, S-VS and P2X7-VS. Pericellular ATP was measured with the pmeLUC probe expressed on the cell surface of untreated CT26 cells or after 5 min of exposure to PBS vehicle, S-VS, and P2X7-VS ( n = 4). H Quantification of luminescence changes was expressed as a fold increase on time 0. I Representative images of photon emissions. Changes in ATP J concentration increase on time 0 in the supernatants of CT26 cells, untreated or treated with PBS vehicle, S-VS, or P2X7-VS, measured with a luciferin/luciferase assay ( n = 3). Changes in adenosine K concentration increase on time 0 in the supernatants of CT26 cells untreated or treated with PBS vehicle, S-VS, P2X7-VS, or P2X7-VS plus 5uM CD73 inhibitor AB680 ( n = 5). * p < 0.05, ** p < 0.001, *** p < 0,0001, **** p < 0.00001.

Article Snippet: Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology).

Techniques: Staining, Confocal Microscopy, Activation Assay, Western Blot, Concentration Assay, Luciferase

Journal: Cell Death & Disease

Article Title: P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: Role of the A2A/CD39/CD73 axis

doi: 10.1038/s41419-025-07897-2

Figure Lengend Snippet: The mRNA expression of P2X7A A , B , P2X7B C , D , CD39 E , F , CD73 G , H , and A2A I , J was evaluated in the cDNAs of 158 patients with CRC subdivided into stage I ( n = 24), stage II ( n = 50), stage III ( n = 52), and stage IV ( n = 32) which comprised 11 samples derived from metastases in organs other than the colon. K Spearman’s correlation coefficient among P2X7A, P2X7B, CD39, CD73 , and A2A was evaluated in CRC metastatic patients. L Spearman’s correlation coefficient was evaluated between P2X7 and A2A expression in colon adenocarcinoma samples obtained from the Cancer Genome Atlas database. * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001.

Article Snippet: Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology).

Techniques: Expressing, Derivative Assay

Journal: Cell Death & Disease

Article Title: P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: Role of the A2A/CD39/CD73 axis

doi: 10.1038/s41419-025-07897-2

Figure Lengend Snippet: mRNA expression of A P2X7A , B P2X7B , C A2A , D CD39 , and E CD73 in CRC patients subdivided into APC WT and APC mutated groups ( n = 6). Percentage of cells positive for P2X7 F and A2A G in the colons of WT and PIRC rats and PIRC tumors ( n = 4). Representative images of immunohistochemical staining for P2X7 and A2A in the colon of WT 1-year rats H, K and in the normal colon I, L and the tumor mass J, M of 1-year PIRC rats. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Orthogonal and Robust Analytics Enable Reproducible and Scalable Manufacturing of High Purity Extracellular Vesicles Derived from Mesenchymal Stromal Cells

doi: 10.1101/2025.08.26.672476

Figure Lengend Snippet: Downstream Processing (DSP) Scalability; Scaled DSP for MSC derived EVs and orthogonal analytical assessment of particle recovery and impurity removal. (a) Schematic representation of the scaled DSP workflow comprising clarification, tangential flow filtration (TFF), ion exchange (IEX) chromatography, post-IEX buffer exchange via TFF, and sterile filtration. Particle recoveries for each unit operation were determined by (b) -NTA, (c) Fl-NTA, and (d) Fl-FC. Protein removal throughout the entire DSP was quantified using (e) BCA assay, while impurity reduction was evaluated by (f) analytical chromatography through analysis of impurity-associated peak area under the curve (AUC). DNA removal was assessed via (g) PicoGreen™ assay. Representative chromatograms of analytical chromatography for DSP steps are shown in (j) , with EV-size peaks (9–14 min) highlighted in red, impurity peaks (14–20 min) in green, and late-eluting impurities (>20 min) in yellow. Final MSC-EVs preparation characterization included (h) scattering and (i) fluorescence NTA size distribution profiles, (k) transmission electron microscopy (TEM) imaging showing characteristic EV cup-shaped morphology, scale bar: 1 µm, and (l) Simple Western confirming EV marker expression (CD81, CD73, Syntenin) and absence of negative markers (calnexin, albumin). (m–o) Fibroblast scratch migration assay demonstrating retained bioactivity of purified MSC-EVs, quantified by cell density recovery within the wound area over 48 hour, supported by qualitative representative images, scale bar: Unless otherwise specified, Multicomparison statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001; ns not significant).

Article Snippet: The following primary antibodies were diluted to the final concentration of 0.1 μg/μL (1:10) in antibody diluent #2 (ProteinSimple, cat#042-203) and used to probe for the proteins of interest: α-CD9 (Cell Signaling, cat#50–173-3388), α-CD81 (Proteintech, cat#MAB46152,), α-Flotillin-1 (SCBT, cat#sc-74566), α-Syntenin-1 (Abcam, cat#ab133267), α-Albumin (R&D systems, cat#MAB1455), α-CD73 (R&D systems, cat#MAB57951), α-CD105 (R&D systems, cat#AF1097) and α-GAPDH (R&D systems, cat#MAB1455).

Techniques: Derivative Assay, Clarification Assay, Filtration, Chromatography, Buffer Exchange, Sterility, BIA-KA, Picogreen Assay, Fluorescence, Transmission Assay, Electron Microscopy, Imaging, Simple Western, Marker, Expressing, Migration, Purification, Comparison

Journal: Journal of tissue engineering

Article Title: Therapeutic potential of epiphyseal growth plate cells for bone regeneration in an osteoporosis model.

doi: 10.1177/20417314221116754

Figure Lengend Snippet: Figure 2. Cellular characterization of EGPCs and BM-MSCs: (a) observation of cell morphology and measurement of cell shape (length to width ratio), (b) the proliferation rates of EGPCs and BM-MSCs were measured from day 1 to day 7 by the CCK-8, (c) cell-cycle analysis of BM-MSCs and EGPCs by using propidium-iodide staining. Quantitation of the cells at each-cycle phase, and (d) Flow-cytometric analyses for the surface markers on BM-MSCs and EGPCs. Representative flow-cytometry profiles are depicted for CD73, CD44, CD90, and CD200. Black and red plots represent the negative control and experimental samples, respectively.

Article Snippet: Antibodies against CD73 (NBP2-25235; 5′-Nucleotidase/CD73 Antibody, NOVUS BIOLOGICALS), CD44 (MCA643GA; MOUSE ANTI RAT CD44, Bio-Rad, UK), CD90 (sc-53456; Thy-1/CD90 antibody (aTHy-1A1), Santa Cruz Biotechnology), and CD200 (MA1-70035; CD200 Monoclonal Antibody (OX-2), ThermoFisher Scientific) were used as the primary antibodies (1:100 diluted in the FACS buffer).

Techniques: CCK-8 Assay, Cell Cycle Assay, Staining, Quantitation Assay, Flow Cytometry, Negative Control