p2x4  (Proteintech)

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Effect of P2X4 receptors in TAMs on the cGAS-STING pathway and mtDNA release (A) PBMC-derived macrophages were induced into TAMs by SW480-conditioned medium with the addition of 20 μmol/L BAY-1797 (P2X4 blocker), 20 μmol/L A-740003 (P2X7 blocker), and 20 μmol/L NF279 (P2X1 blocker), respectively. An equal final concentration of DMSO (0.05%) was used including vehicle controls. The STAT1 phosphorylation were observed by western blot. (B) P2X4 receptor expression in M1/M2 macrophages was observed by cytometry. Median fluorescence intensity (MFI) was compared (∗∗ p < 0.01, One-way ANOVA). (C) Lentiviral transfection was performed to construct P2X4 overexpression or knockdown stable cell lines of THP-1 cells, the expression of P2X4 protein was verified. The differences in protein phosphorylation were compared among TAMs derived from each stable cell line by western blotting. (D) The surface markers of macrophage (CD80-APC, CD163-PE) were compared by cytometry (∗∗ p < 0.01, One-way ANOVA). (E) The mRNA levels of M1 marker genes and cytokine secretion levels were compared via qPCR and ELISA (∗∗ p < 0.01, Kruskal-Wallis test). (F) The mRNA levels of M2 marker genes were compared via qPCR (Kruskal-Wallis test). All data are presented as mean ± SD. Data points represent independent biological replicates. Western blots images shown are representative of 3 independent experiments.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Derivative Assay, Concentration Assay, Phospho-proteomics, Western Blot, Expressing, Cytometry, Fluorescence, Transfection, Construct, Over Expression, Knockdown, Stable Transfection, Marker, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Mechanism of mitochondrial calcium overload by P2X4 receptor-mediated calcium influx in TAMs (A) TAMs were induced from THP-1-derived macrophages. Cytosolic dsDNA was detected using an anti-dsDNA antibody with Alexa Fluor 488 (green), and nuclei were counterstained with DAPI (blue). Images were acquired by confocal microscopy (scale bars, 10 μm). Representative image shown ( N = 3 independent experiments, 10 images were analyzed per experiment). The boxplot shows the signal intensity (∗∗ p < 0.01, Student’s t test). (B) BAY-1797 was added during induction of TAMs. The trends of mtDNA/nDNA changes in the cytoplasm of cells were compared by detecting reference genes via qPCR (∗∗ p < 0.01, Student’s t test). (C) After incubation THP-1-derived macrophages from P2X4-OE, shP2X4, and control groups with the Ca 2+ probe Rhod-2, the magnitude of Ca 2+ concentration fluctuations in response to 100 μM eATP stimulation was measured on a fluorometric plate reader. (D) The THP-1 stable cell lines were induced into TAMs and Ca 2+ fluorescence (scale bars, 10 μm) was observed under a confocal microscope ( N = 3 independent experiments). (E) THP-1-derived macrophages (5 biological samples per group) were loaded with Rhod-2 probe, mitoSOX probe or JC-1 probe, respectively. After 3 h of induction by adding SW480-conditioned medium, intracellular Ca 2+ concentration, mitochondrial ROS levels and mitochondrial membrane potential changes were detected. All experiments were independently performed three times. (F) During induction of TAM, simultaneous NAC intervention was set up or calcium-free SW480-conditioned medium was used. Intracellular Ca 2+ concentration and mitochondrial ROS levels were detected. Differences in the mtDNA/nDNA ratio in the cytoplasm were compared (∗ p < 0.05, ∗∗ p < 0.01, One-way ANOVA). (G) After TAM induction from THP-1-derived macrophages stably overexpressing FLAG-cGAS, cGAS ChIP was performed and mtDNA/nDNA enrichment was compared by detecting reference genes via qPCR (∗∗ p < 0.01, Student’s t test). All data are presented as mean ± SD. Data points represent independent biological replicates.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Derivative Assay, Confocal Microscopy, Incubation, Control, Concentration Assay, Stable Transfection, Fluorescence, Microscopy, Membrane

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Effects of P2X4 knockout on the polarization of TAMs and T cell regulation (A) P2X4 knockout Ana-1 cell lines were constructed. western blot detected P2X4 expression in three of the monoclonal cell lines. Western blots shown are representative of 3 independent experiments. (B) Wild type or P2X4-K.O. Ana-1 cells were induced into TAMs by MC38 conditioned medium. The mRNA expression of M1/M2 cytokines were compared by qPCR 48 h later (∗ p < 0.05, ∗∗ p < 0.01, One-way ANOVA). (C) Suspension mononuclear cells isolated from the spleen of C57BL/6J mice were grouped to be co-cultured with TAMs (sgP2X4) or TAMs (WT). Living cells were counted using a cell viability analyzer at time points (∗∗ p < 0.01, Student’s t test). (D) After 72 h, the suspension cells were harvested and stained with CD3 (PE-Cy7), CD4 (PE), CD8 (FITC), and CXCR6 (APC) for flow cytometry. Another set of cells were fixed for GzmB (BV421) staining after stimulated with a combination of 50 ng/mL PMA, 1 μg/mL ionomycin, and 5 μg/mL brefeldin A for 4 h. Data were compared by histogram (∗ p < 0.05, ∗∗ p < 0.01, Student’s t test). All data are presented as mean ± SD. Data points represent independent biological replicates.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Knock-Out, Construct, Western Blot, Expressing, Suspension, Isolation, Cell Culture, Staining, Flow Cytometry

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Effect of P2X4 knockout macrophages on T cell infiltration in MC38 tumors in mice (A) 6-week C57BL/6J female mice were divided into 3 groups and inoculated as described. Tumor volume and weight were compared (∗ p < 0.05, ∗∗ p < 0.01, Student’s t test). (B) Subcutaneous tumors in each group (4 per group, randomly selected) were digested into single-cell suspensions and stained with antibodies against CD45 (PE-Cy7), F4/80 (AF647), CD80 (PE-CF594), and CD86 (PE) for macrophages, or with antibodies against CD3 (PE-Cy7), CD4 (PE), CD8 (FITC), CXCR6 (APC) and GzmB (BV421) for T cells before flow cytometry. (C) The total number of tumor infiltrating immune cells was measured and the proportions of TAMs and T cells in each group were compared by histogram. (∗ p < 0.05, ∗∗ p < 0.01, Student’s t test). All data are presented as mean ± SD. Data points represent independent biological replicates.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Knock-Out, Staining, Flow Cytometry

Journal: iScience

Article Title: M1-like macrophages regulate T cell infiltration in colorectal cancer through P2X4 receptor

doi: 10.1016/j.isci.2025.113517

Figure Lengend Snippet: Characterization of P2X4 receptor expression and prognosis in CRC (A) P2X4 protein expression in tumor tissue (T) and adjacent normal tissue (N) from 24 CRC patients was detected by western blot. Relative P2X4 expression were quantified by densitometry and compared in a paired dot plot (∗∗ p < 0.01, paired Student’s t test). (B) Immunofluorescence staining (P2X4-AF488, CD68-AF594) was performed and observed by confocal microscopy (scale bars, 50 μm). Representative images shown from paired CRC and adjacent tissues ( n = 12 patients). Boxplot below indicates the number of P2X4 + CD68 + cells per field of view (∗∗ p < 0.01, Student’s t test). (C) 457 COAD patients in the TCGA database were divided into P2X4 high and low expression groups using FPKM = 3.67 as a cut-off, and overall survival was compared ( p = 0.014, Log rank test). (D) Correlation between P2X4 and CXCR6 expression in 623 CRC patients from TCGA database (R = 0.31, p < 0.01, Spearman Analysis). Data are presented as mean ± SD. Each dot represents one individual patient sample.

Article Snippet: FITC anti-human P2X4 , alomone labs (Israeli) , Cat# APR024-F.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Confocal Microscopy

Journal: Journal of Innate Immunity

Article Title: Potential Role of Aerobic Exercise in Attenuating Diabetic Cardiomyopathy via Modulation of P2X4-Mediated NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1159/000548603

Figure Lengend Snippet: Exercise training suppressed PANX1/P2X4/NLRP3 signaling and reduced myocardial oxidative stress. a A PPI network of NLRP3-related protein and purinergic signaling-related protein. b qRT-PCR analysis of PANX1 mRNA levels among different groups, n = 9. c qRT-PCR analysis of relative mRNA expression of P2X1–7 to GAPDH (housekeeping) in the heart of normal mice, n = 4. d qRT-PCR analysis of P2X1, P2X4, P2X5, P2X7 mRNA levels among different groups, n = 6–9. e Representative WB images of PANX1, P2X4, P2X7, TXNIP, NLRP3 protein, n = 9. f , g qRT-PCR analysis of TXNIP and NFE2L2 mRNA levels among different groups, n = 6–9. h The MDA levels among different groups, n = 4. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM. See also online supplementary Figure S1. SED, sedentary; EX, exercise; DM, diabetes mellitus; qRT-PCR, quantitative real-time-PCR; PPI, protein-protein interaction.

Article Snippet: The membranes were then blocked in QuickBlockTM Blocking Buffer for Western Blot (Beyotime, P0252, China) for 2 h at room temperature and then incubated with primary antibodies directed against PANX1 (12595-1-AP, Proteintech, China), IL-1β (sc-12742, Santa Cruz, USA), P2X7 (A10511, ABclonal, China), caspase-1 (A0964, ABclonal, China), GSDMD (BS-14287R, Bioss, China), P2X4 (66416-1-Ig, Proteintech, China), NLRP3 (DF7438, affinity, China), TXNIP (sc-166234, Santa, USA), NF-κb p65 (WL01980, Wanleibio, China), p-NF-κb (WL02169, Wanleibio, China), β-tubulin (AF7011, affinity, China), HSP90 (ab13492, Abcam, USA), and GAPDH (sc-47724, Santa, USA) and horseradish peroxidase-conjugated secondary antibodies (115-035-003, 111-035-003, Jackson Lab, USA).

Techniques: Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Innate Immunity

Article Title: Potential Role of Aerobic Exercise in Attenuating Diabetic Cardiomyopathy via Modulation of P2X4-Mediated NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1159/000548603

Figure Lengend Snippet: Hyperlipid-induced P2X4 upregulation and NLRP3 inflammasome activation. a The viability of H9C2 cells treated with high Glu/high PA; experiments were performed in 4 biological replicates with 3 technical replicates. b LDH content in the supernatant of H9C2 cells treated with high Glu/high PA. Experiments were performed in 4 biological replicates with 3 technical replicates. c Representative WB images of PANX1, NLRP3, P2X7, P2X4 protein, n = 4–6. d Caspase-1 activity of H9C2 cells treated with high Glu/high PA; all experiments were performed in 4 biological replicates. e The viability of H9C2 cells treated with high Glu/high PA, MCC950, KCl, and CaCl 2 ; experiments were performed in 3 biological replicates with 3 technical replicates. f The viability of H9C2 cells treated with KCl; experiments were performed in 3 biological replicates with 3 technical replicates. g Representative images of H9C2 stimulated with high Glu/high PA and stained with ROS dye. White arrowheads indicate membrane rupture and ballooning (pyroptosis), while black arrowheads indicate cell shrinkage and apoptotic body formation (apoptosis). Scale bar, 100 μm, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the CON group (5.5 m m glucose) or comparison as indicated. Data are represented as mean ± SEM. See also online supplementary Figure S2. Glu, glucose; PA, palmitic acid; LDH, lactate dehydrogenase; blank, no treatment; Con, mannitol and BSA treatment as control; CON, normal diet mice; HFD, high-fat diet mice; HE, HFD and exercise mice.

Article Snippet: The membranes were then blocked in QuickBlockTM Blocking Buffer for Western Blot (Beyotime, P0252, China) for 2 h at room temperature and then incubated with primary antibodies directed against PANX1 (12595-1-AP, Proteintech, China), IL-1β (sc-12742, Santa Cruz, USA), P2X7 (A10511, ABclonal, China), caspase-1 (A0964, ABclonal, China), GSDMD (BS-14287R, Bioss, China), P2X4 (66416-1-Ig, Proteintech, China), NLRP3 (DF7438, affinity, China), TXNIP (sc-166234, Santa, USA), NF-κb p65 (WL01980, Wanleibio, China), p-NF-κb (WL02169, Wanleibio, China), β-tubulin (AF7011, affinity, China), HSP90 (ab13492, Abcam, USA), and GAPDH (sc-47724, Santa, USA) and horseradish peroxidase-conjugated secondary antibodies (115-035-003, 111-035-003, Jackson Lab, USA).

Techniques: Activation Assay, Activity Assay, Staining, Membrane, Comparison, Control

Journal: Journal of Innate Immunity

Article Title: Potential Role of Aerobic Exercise in Attenuating Diabetic Cardiomyopathy via Modulation of P2X4-Mediated NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1159/000548603

Figure Lengend Snippet: P2X4 and P2X7 were upregulated in the hearts of HFD mice and restrained by exercise intervention. a Representative M-mode echocardiograms of the LVs are shown. b–f Quantification of corrected LV mass ( b ), LVEF ( c ), LVFS values ( d ), LVIDs ( e ), and LVIDd ( f ) among different groups, n = 5. g Representative WB images of PANX1, P2X7, P2X4, NLRP3, caspase-1 protein, n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM. See also online supplementary Figures S3 and S4. CON, normal diet mice; HFD, high-fat diet mice; HE, HFD and exercise mice.

Article Snippet: The membranes were then blocked in QuickBlockTM Blocking Buffer for Western Blot (Beyotime, P0252, China) for 2 h at room temperature and then incubated with primary antibodies directed against PANX1 (12595-1-AP, Proteintech, China), IL-1β (sc-12742, Santa Cruz, USA), P2X7 (A10511, ABclonal, China), caspase-1 (A0964, ABclonal, China), GSDMD (BS-14287R, Bioss, China), P2X4 (66416-1-Ig, Proteintech, China), NLRP3 (DF7438, affinity, China), TXNIP (sc-166234, Santa, USA), NF-κb p65 (WL01980, Wanleibio, China), p-NF-κb (WL02169, Wanleibio, China), β-tubulin (AF7011, affinity, China), HSP90 (ab13492, Abcam, USA), and GAPDH (sc-47724, Santa, USA) and horseradish peroxidase-conjugated secondary antibodies (115-035-003, 111-035-003, Jackson Lab, USA).

Techniques:

Journal: Journal of Innate Immunity

Article Title: Potential Role of Aerobic Exercise in Attenuating Diabetic Cardiomyopathy via Modulation of P2X4-Mediated NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1159/000548603

Figure Lengend Snippet: AICAR downregulated P2X4 and suppressed NLRP3 inflammasome activation of high-glucose/high PA-induced cardiomyocytes. a The viability of H9C2 cells treated with AICAR and high Glu/high PA; experiments were performed in 4 biological replicates with 3 technical replicates. b LDH content in the supernatant of H9C2 cells treated with AICAR and high Glu/high PA; experiments were performed in 4 biological replicates with 3 technical replicates. c Representative WB images of NLRP3, P2X7, P2X4 protein; all experiments were performed in at least 4 biological replicates. d The caspase-1 activity of H9C2 cells treated with AICAR and high Glu/high PA; experiments were performed in 4 biological replicates with 3 technical replicates. # p < 0.05, ### p < 0.001 versus the Con group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus the Glu + PA group (25 m m Glu + 300 μ m PA). Data are represented as mean ± SEM. Glu, glucose; PA, palmitic acid; LDH, lactate dehydrogenase; Con, mannitol and BSA treatment as control.

Article Snippet: The membranes were then blocked in QuickBlockTM Blocking Buffer for Western Blot (Beyotime, P0252, China) for 2 h at room temperature and then incubated with primary antibodies directed against PANX1 (12595-1-AP, Proteintech, China), IL-1β (sc-12742, Santa Cruz, USA), P2X7 (A10511, ABclonal, China), caspase-1 (A0964, ABclonal, China), GSDMD (BS-14287R, Bioss, China), P2X4 (66416-1-Ig, Proteintech, China), NLRP3 (DF7438, affinity, China), TXNIP (sc-166234, Santa, USA), NF-κb p65 (WL01980, Wanleibio, China), p-NF-κb (WL02169, Wanleibio, China), β-tubulin (AF7011, affinity, China), HSP90 (ab13492, Abcam, USA), and GAPDH (sc-47724, Santa, USA) and horseradish peroxidase-conjugated secondary antibodies (115-035-003, 111-035-003, Jackson Lab, USA).

Techniques: Activation Assay, Activity Assay, Control

Journal: Journal of Innate Immunity

Article Title: Potential Role of Aerobic Exercise in Attenuating Diabetic Cardiomyopathy via Modulation of P2X4-Mediated NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1159/000548603

Figure Lengend Snippet: Downregulated P2X4 and P2X7 suppressed high-glucose/high PA-induced NLRP3 expression. a–c qRT-PCR analysis of P2X7, P2X4, and NLRP3 mRNA levels among different groups; all experiments were performed in at least 4 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 versus siNC group, # p < 0.05 compared to the Glu + PA group (25 m m glucose + 300 μ m PA). Data are represented as mean ± SEM. qRT-PCR, quantitative real-time-PCR.

Article Snippet: The membranes were then blocked in QuickBlockTM Blocking Buffer for Western Blot (Beyotime, P0252, China) for 2 h at room temperature and then incubated with primary antibodies directed against PANX1 (12595-1-AP, Proteintech, China), IL-1β (sc-12742, Santa Cruz, USA), P2X7 (A10511, ABclonal, China), caspase-1 (A0964, ABclonal, China), GSDMD (BS-14287R, Bioss, China), P2X4 (66416-1-Ig, Proteintech, China), NLRP3 (DF7438, affinity, China), TXNIP (sc-166234, Santa, USA), NF-κb p65 (WL01980, Wanleibio, China), p-NF-κb (WL02169, Wanleibio, China), β-tubulin (AF7011, affinity, China), HSP90 (ab13492, Abcam, USA), and GAPDH (sc-47724, Santa, USA) and horseradish peroxidase-conjugated secondary antibodies (115-035-003, 111-035-003, Jackson Lab, USA).

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Journal of Innate Immunity

Article Title: Potential Role of Aerobic Exercise in Attenuating Diabetic Cardiomyopathy via Modulation of P2X4-Mediated NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1159/000548603

Figure Lengend Snippet: AICAR treatment and downregulated P2X4 and P2X7 suppressed high-glucose/high PA-induced pyroptosis and oxidative stress. a Representative IF pictures of GSDMD, TUNEL, DAPI, ROS, scale bar, 100 μm. b Statistical graphs of GSDMD and TUNEL double-positive ratio analysis among different groups were performed. c Statistical graphs of ROS production analysis among different groups were performed. d NOX activity among different groups. e The IL-1β levels in cell supernatant among different groups. All experiments were performed with 4 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM. Potential role of aerobic exercise in attenuating DCM via modulation of P2X4-mediated NLRP3 inflammasome activation and pyroptosis. Glu, glucose; PA, palmitic acid; LDH, lactate dehydrogenase; NOX, NADPH oxidase; Sup, supernatant.

Article Snippet: The membranes were then blocked in QuickBlockTM Blocking Buffer for Western Blot (Beyotime, P0252, China) for 2 h at room temperature and then incubated with primary antibodies directed against PANX1 (12595-1-AP, Proteintech, China), IL-1β (sc-12742, Santa Cruz, USA), P2X7 (A10511, ABclonal, China), caspase-1 (A0964, ABclonal, China), GSDMD (BS-14287R, Bioss, China), P2X4 (66416-1-Ig, Proteintech, China), NLRP3 (DF7438, affinity, China), TXNIP (sc-166234, Santa, USA), NF-κb p65 (WL01980, Wanleibio, China), p-NF-κb (WL02169, Wanleibio, China), β-tubulin (AF7011, affinity, China), HSP90 (ab13492, Abcam, USA), and GAPDH (sc-47724, Santa, USA) and horseradish peroxidase-conjugated secondary antibodies (115-035-003, 111-035-003, Jackson Lab, USA).

Techniques: TUNEL Assay, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Membrane Proteins at Scale: Automated Copolymer Nanodisc Purification for Structure and Function

doi: 10.1101/2025.09.05.674548

Figure Lengend Snippet: a. + d. Solubilization and purification efficiency of 32 different copolymers was screened for P2X4 Tni and P2X4 HEK. . SDS–PAGEs of the eluates were analyzed either by densitometry (ImageJ Gel Analyzer) (b,e) or by buried tryptophan fluorescence at 330 nm (NanoTemper Panta Discovery) (c,f). Intensities were normalized to the strongest signal in each dataset (set to 100%) and plotted as box plots to visualize distributions across copolymer families. Corresponding WBs can be viewed in Fig. S6.

Article Snippet: Binding kinetics of 5-BDBD to P2X4 were assessed using grating-coupled interferometry (GCI, WAVEsystem, Creoptix).

Techniques: Purification, Fluorescence

Journal: bioRxiv

Article Title: Membrane Proteins at Scale: Automated Copolymer Nanodisc Purification for Structure and Function

doi: 10.1101/2025.09.05.674548

Figure Lengend Snippet: a. Dynamic light scattering (DLS) of P2X4 Tni solubilized in SMA200, AASTY 6-55, and Glyco-Cubipol revealed heterogeneous particle populations with large hydrodynamic radii (hdR) and high PDIs. b. NanoDSF of the same P2X4 Tni samples showed no detectable inflection points (IPs), consistent with conformational heterogeneity. c. DLS of P2X4 HEK solubilized in SMA200, AASTY 6-55, and Glyco-Cubipol demonstrated compact and homogeneous nanodiscs with hdR values of 7.3 nm (PDI 0.50), 8.5 nm (PDI 0.10), and 7.9 nm (PDI 0.29), respectively. d. NanoDSF of P2X4 HEK revealed distinct IPs at 50.9 °C (SMA200), 56.7 °C (AASTY 6-55), and 65 °C (Glyco-Cubipol). e. Copolymer screening of full-length P2X4 expressed in HEK293 cells. Thirty-two copolymers representing six backbones were tested for solubilization and stabilization efficiency. Following purification, eluates were analyzed by Coomassie-stained gels for yield and by biophysical methods including T m , hdR, PDI, and turbidity. Values were normalized and integrated into a composite score (T m 0.35, hdR 0.25, PDI 0.10, turbidity 0.05, elution quantity 0.25). Copolymers were ranked by this score, with lower ranks indicating superior performance. The scatter plot displays T mIP (x-axis) versus hdR (y-axis), visualizing stability and size characteristics. Top performers included several Cubipol derivatives (e.g., Glyco-Cubipol, Sulfo-Cubipol Medium, Cubipol PEG, Cubipol Amine; shown in green), which consistently combined high thermostability with favorable size distribution and yield. The full dataset is provided in Figure S9 + S7,8.

Article Snippet: Binding kinetics of 5-BDBD to P2X4 were assessed using grating-coupled interferometry (GCI, WAVEsystem, Creoptix).

Techniques: Nano Differential Scanning Fluorimetry, Purification, Staining

Journal: bioRxiv

Article Title: Membrane Proteins at Scale: Automated Copolymer Nanodisc Purification for Structure and Function

doi: 10.1101/2025.09.05.674548

Figure Lengend Snippet: a. Anti-rho western blot and SDS-PAGE analysis of the PNGase digestion of P2X4 HEK . A clear shift of the P2X4 bands before (1) and after (2) digestion demonstrates that the higher apparent molecule weight is due to glycosylation of the protein. b . Native PAGE showing that P2X4 HEK is assembled as a trimer. c . Grating-coupled interferometry (GCI) sensorgrams of BAY-1797 binding to P2X4 HEK reconstituted in U18 biotin nanodiscs. Serial twofold dilutions (39 nM–20 µM) were injected at 25°C onto immobilized P2X4–U18 biotin nanodiscs. Sensorgrams were blank/DMSO corrected and globally fitted to a 1:1 Langmuir binding model, yielding a dissociation constant (Kd) of 1.3 µM with kinetic rate constants k on = 9.28 ± 3.03 x 10 3 M -1 S -1 and k off = 1.15 ± 0.30 × 10 -1 S -1 d. Spectral shift analysis of BAY-1797 binding to fluorescently labelled full-length P2X4 HEK in U18 biotin nanodiscs. Fluorescence shifts (ΔRatio 670/650 nm) were recorded upon titration of BAY-1797 (twofold serial dilution) and fitted by nonlinear regression, yielding a K d of 15.6 µM. e. GCI WaveRAPID analysis of 5-BDBD binding to P2X4 HEK –U18 biotin nanodiscs. Sensorgrams were referenced against empty nanodisc controls and fitted to a conformational-change binding model, giving a K d of ∼10 µM. f. Spectral shift assay of 5-BDBD with P2X4 HEK in U18 biotin nanodiscs. Titration of fluorescently labelled receptor with serial dilutions of ligand produced concentration-dependent fluorescence shifts, yielding a binding affinity of 5.4 µM.

Article Snippet: Binding kinetics of 5-BDBD to P2X4 were assessed using grating-coupled interferometry (GCI, WAVEsystem, Creoptix).

Techniques: Western Blot, SDS Page, Glycoproteomics, Clear Native PAGE, Binding Assay, Injection, Fluorescence, Titration, Serial Dilution, Shift Assay, Produced, Concentration Assay

Journal: bioRxiv

Article Title: Membrane Proteins at Scale: Automated Copolymer Nanodisc Purification for Structure and Function

doi: 10.1101/2025.09.05.674548

Figure Lengend Snippet: a . Representative 2D classes obtained from the data sets of P2X4T.ni in Glyco-DIBMA, Ultrasolute-18, Glyco-Cubipol, and Cubipol Glycerol. Glyco-DIBMA shows almost absolute preferential orientation. For all other polymers, a reconstruction together with an angular distribution histogram is shown next to the 2D classes. b . P2X4 T.ni in Sulfo-Cubipol was chosen for a large Cryo-EM data set. As above, 2D classes and angular distribution plots are shown on the left. High-resolution cryo-EM reconstructions of Apo P2X4 T.ni or in complex with ATP/5-BDBD are shown on the right. Density corresponding for ATP is highlighted in magenta, and shown in more detail in the close-up panel.

Article Snippet: Binding kinetics of 5-BDBD to P2X4 were assessed using grating-coupled interferometry (GCI, WAVEsystem, Creoptix).

Techniques: Cryo-EM Sample Prep