Journal: PLoS ONE

Article Title: Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220 + Double-Negative T Lymphocytes of Autoimmune MRL/ lpr Mice

doi: 10.1371/journal.pone.0052161

Figure Lengend Snippet: Spleen cells from 3 to 4-mo-old MRL +/+ ( solid line ), MRL/ lpr ( dashed line ) and P2X7-deficient B6 P2X7R−/− ( dotted line ) mice (n = 3 mice/group) were treated for 45 min at 37°C with doses of ATP ranging from 100 to 5000 µM. Spleen cells were then triple-stained with anti-CD90, anti-CD19 and anti-CD62L mAb to assess by flow cytometry: (A) the percentage of CD62L-expressing CD19 – CD90 + T cells and (B) MFI of CD62L on CD19 – CD90 + T cells. Results are expressed as the mean percentage of initial expression ± SE.

Article Snippet: The membranes were blocked in TBS containing 3% nonfat dry milk and 0.2% Tween 20 for 1 h at room temperature and subsequently incubated overnight at 4°C with affinity purified rabbit anti-P2X7R antibodies recognizing residues 576 to 595 of P2X7R (1∶1000; Alomone Laboratories, Jerusalem, Israel).

Techniques: Staining, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220 + Double-Negative T Lymphocytes of Autoimmune MRL/ lpr Mice

doi: 10.1371/journal.pone.0052161

Figure Lengend Snippet: (A) The levels of CD39 expression on B220 – (either CD4 + or CD8 + ) (□) and B220 + DN (▪) T-cell subsets as well as on CD19 + B cells (▪) from MRL/ lpr mice (n = 3) were analyzed by flow cytometry. Results shown on histogram are normalized MFI (nMFI) calculated as MFI of positive cells × percentage of positive cells. Asterisks denote statistically significant differences between B220 + DN T cells or B220 – CD8 + T cells and B220 – CD4 + T cells: * p ≤0.05; ** p ≤0.01. (B) P2X7R mRNA expression was analyzed in FACS-purified B220 – (□) and B220 + (▪) CD19 – CD90 + T-cell subsets from individual B6, MRL +/+ and MRL/ lpr mice by real-time RT-PCR. Specific P2X7R cDNA was quantified by standard curves based on known amounts of PCR-amplified P2X7R cDNA. Data are expressed as amount (pg) of P2X7R RNA per cell, and histograms represent the mean ± SE of three independent experiments (n = 8 mice/group). (C) P2X7R protein levels in whole LN cells from 3 to 4-mo-old MRL +/+ , MRL/ lpr and B6 P2X7−/− mice, and FACS-sorted B220 – and B220 + CD19 – CD90 + T-cell subsets from MRL/ lpr mice were analyzed by western blotting using affinity-purified rabbit anti-P2X7R polyclonal antibodies (1∶1000; Alomone Laboratories). A non-specific band (*) is frequently observed with this polyclonal antibody . The blot was stripped and reprobed with anti-actin mAb. (D and E) Flow cytometric analysis of P2X7R expression levels on B220 – and B220 + T-cell subpopulations. Spleen cells from MRL +/+ , MRL/ lpr and B6 P2X7R−/− mice (n = 3 mice/group) were stained with anti-CD90 mAb, anti-B220 mAb, rabbit polyclonal anti-P2X7R antiserum (1∶100) generated by genetic immunization and with either anti-CD4 mAb, anti-CD8 mAb or anti-CD4 plus anti-CD8 mAbs. (D) Overlay histograms showing the expression levels of P2X7R on gated CD4 T cells or CD8 T cells from MRL +/+ mice (open histogram) and B6 P2X7R−/− mice (shaded histogram). (E) Overlay histograms comparing the expression levels of P2X7R on B220 – (–) and B220 + (–) T cells, either CD4 + ( left ) or CD8 + ( middle ), from MRL/ lpr mice. Right , overlay histogram comparing the expression levels of P2X7R on B220 + DN T cells (–) and B220 – CD4 + T cells (–) from MRL/ lpr mice. The results shown are representative of at least four independent experiments.

Article Snippet: The membranes were blocked in TBS containing 3% nonfat dry milk and 0.2% Tween 20 for 1 h at room temperature and subsequently incubated overnight at 4°C with affinity purified rabbit anti-P2X7R antibodies recognizing residues 576 to 595 of P2X7R (1∶1000; Alomone Laboratories, Jerusalem, Israel).

Techniques: Expressing, Flow Cytometry, Purification, Quantitative RT-PCR, Amplification, Western Blot, Affinity Purification, Staining, Generated

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: The sequences of oligonucleotide primers.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques:

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: The antibodies for immunoblotting.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Western Blot, Concentration Assay

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: The antibodies for fluorescence labeling.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Fluorescence, Labeling, Concentration Assay

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Injection, Western Blot, Immunofluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Immunofluorescence, Labeling, Fluorescence

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: IL-1 β , IL-6, and TNF- α mRNA expression in DRG and SDH after inhibition of P2X7R. (a) DRG IL-1 β mRNA. (b) DRG IL-6 mRNA. (c) DRG TNF- α mRNA. (d) SDH IL-1 β mRNA. (e) SDH IL-6 mRNA. (f) SDH TNF- α mRNA. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38. Mean ± SEM ( n = 5). ∗∗∗ P < 0.001 (vs. control); # P < 0.05; ## P < 0.01 (vs. BTZ group).

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Inhibition

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: P2X7 Receptor (P2X7R) of Microglia Mediates Neuroinflammation by Regulating (NOD)-Like Receptor Protein 3 (NLRP3) Inflammasome-Dependent Inflammation After Spinal Cord Injury

doi: 10.12659/MSM.925491

Figure Lengend Snippet: Immunofluorescence detected P2X7R of microglia in spinal cord. ( A ) P2X7R (P2X7R+, green) and IBA-1 (IBA-1+, red) co-expressed as P2X7R of microglia (P2X7R+/IBA-1+, yellow) that were overexpressed after spinal cord injury and BzATP intervention, and, compared to the morphology of microglia in the sham group, the morphology of activated microglia after injury changed to large soma and short irregular processes, revealing that many microglia in spinal cord became active after spinal cord injury. ( B ) The number of P2X7R of microglia (P2X7R+/IBA-1+) increased notably after spinal cord injury compared to the sham group (** P<0.01), and, compared to the model group, BzATP increased the number of P2X7R of microglia (* P<0.05) while A-438079 significantly decreased the number of P2X7R of microglia (** P<0.01). ( C ) The P2X7R-positive microglia percentage was clearly increased after spinal cord injury compared to the sham group (** P<0.01), and, compared to the model group, BzATP accounted for a significantly increased P2X7R-positive microglia proportion (** P<0.01) while A-438079 accounted for a significantly reduced P2X7R-positive microglia proportion (** P<0.01).

Article Snippet: Sections were subjected to antigen retrieval with citric acid buffer, blocked with 10% normal goat serum for 2 h at room temperature, and incubated with a mixture of rabbit P2X7R antibody (1: 100, APR-004, Alomone, Jerusalem, Israel) and goat IBA-1 antibody (1: 200, ab5076, Abcam, Shanghai, China) at 4°C overnight.

Techniques: Immunofluorescence

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: P2X7 Receptor (P2X7R) of Microglia Mediates Neuroinflammation by Regulating (NOD)-Like Receptor Protein 3 (NLRP3) Inflammasome-Dependent Inflammation After Spinal Cord Injury

doi: 10.12659/MSM.925491

Figure Lengend Snippet: Western blot and qPCR assay were used to assess the expression of P2X7R protein and P2X7R mRNA in spinal cord. ( A ) Western blot assay showed that the expression of P2X7R protein in spinal cord was increased notably after spinal cord injury compared to the sham group (** P<0.01) and, compared to model group, BzATP increased the expression of P2X7R protein in spinal cord (* P<0.05) while A-438079 significantly decreased the expression of P2X7R protein in spinal cord (** P<0.01). ( B ) qPCR assay showed that the expression of P2X7R mRNA in spinal cord increased notably after spinal cord injury compared to the sham group (** P<0.01), and compared to the model group, BzATP increased the expression of P2X7R mRNA in spinal cord (* P<0.05) while A-438079 significantly decreased the expression of P2X7R mRNA in spinal cord (** P<0.01).

Article Snippet: Sections were subjected to antigen retrieval with citric acid buffer, blocked with 10% normal goat serum for 2 h at room temperature, and incubated with a mixture of rabbit P2X7R antibody (1: 100, APR-004, Alomone, Jerusalem, Israel) and goat IBA-1 antibody (1: 200, ab5076, Abcam, Shanghai, China) at 4°C overnight.

Techniques: Western Blot, Expressing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: P2X7 Receptor (P2X7R) of Microglia Mediates Neuroinflammation by Regulating (NOD)-Like Receptor Protein 3 (NLRP3) Inflammasome-Dependent Inflammation After Spinal Cord Injury

doi: 10.12659/MSM.925491

Figure Lengend Snippet: P2X7R of microglia in spinal cord-mediated neuroinflammation after spinal cord injury. ( A ) ELISA showed that, compared to the sham group, the expression of IL-1β and IL-18 in the spinal cord were significantly increased after spinal cord injury (** P<0.01) and, compared to the model group, A438079 significantly inhibited the expression of IL- 1β and IL-18 (** P<0.01) and BzATP significantly increased the expression of IL- 1β and IL-18 (** P<0.01), which was strongly inhibited by CY-09 (** P<0.01). ( B ) qPCR assay showed that, compared to the sham group, the expression of IL-1β mRNA and IL-18 mRNA in the spinal cord was significantly increased after spinal cord injury (** P<0.01) and, compared to the model group, A438079 significantly inhibited the expression of IL-1β mRNA and IL-18 mRNA (** P<0.01), and BzATP significantly increased the expression of IL-1β mRNA and IL-18 mRNA (** P<0.01), which was strongly inhibited by CY-09 (** P<0.01).

Article Snippet: Sections were subjected to antigen retrieval with citric acid buffer, blocked with 10% normal goat serum for 2 h at room temperature, and incubated with a mixture of rabbit P2X7R antibody (1: 100, APR-004, Alomone, Jerusalem, Israel) and goat IBA-1 antibody (1: 200, ab5076, Abcam, Shanghai, China) at 4°C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: P2X7 Receptor (P2X7R) of Microglia Mediates Neuroinflammation by Regulating (NOD)-Like Receptor Protein 3 (NLRP3) Inflammasome-Dependent Inflammation After Spinal Cord Injury

doi: 10.12659/MSM.925491

Figure Lengend Snippet: P2X7R of microglia in spinal cord regulated the expression of NLRP3 and cleaved-Caspase-1 (P20). Compared to the sham group, the expression of NLRP3 and cleaved-Caspase-1 (P20) in the spinal cord significantly increased after spinal cord injury (** P<0.01) and, compared to the model group, BzATP promoted the expression of NLRP3 and cleaved-Caspase-1 (P20) (* P<0.05), which was inhibited by CY-09 (* P<0.05), while A-438079 notably reduced the expression of NLRP3 and cleaved-Caspase-1 (P20) (** P<0.01).

Article Snippet: Sections were subjected to antigen retrieval with citric acid buffer, blocked with 10% normal goat serum for 2 h at room temperature, and incubated with a mixture of rabbit P2X7R antibody (1: 100, APR-004, Alomone, Jerusalem, Israel) and goat IBA-1 antibody (1: 200, ab5076, Abcam, Shanghai, China) at 4°C overnight.

Techniques: Expressing

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Primer sequences for PCR analysis in animal experiments.

Article Snippet: Rabbit anti-P2X7R pAb (Alomone) , Ditto.

Techniques: Sequencing

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Primer sequences for PCR analysis in cellular experiments.

Article Snippet: Rabbit anti-P2X7R pAb (Alomone) , Ditto.

Techniques: Sequencing

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Primary and secondary antibodies for Western blot assays.

Article Snippet: Rabbit anti-P2X7R pAb (Alomone) , Ditto.

Techniques: Western Blot

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Effects of ACOS on the expression of P2X7R/NLRP3 inflammasome in the rat DN model. (a) Total RNA was extracted from renal cortex tissue, and the relative mRNA expression levels of P2X7R, NLRP3, ASC, and procaspase-1 were measured by real-time quantitative PCR. (b) Renal cortex tissue was lysed, and total lysates were analyzed by the Western blot assay with antibodies against P2X7R, NLRP3, ASC, active caspase-1 subunit P10, and β -actin, respectively. The relative protein expression level was expressed as the target protein/ β -actin ratio. Values are represented as mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01 vs. control group, # P < 0.05 and ## P < 0.01 vs. DN model group.

Article Snippet: Rabbit anti-P2X7R pAb (Alomone) , Ditto.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Double immunofluorescence staining of the podocyte marker and P2X7R/NLRP3 in renal tissues of the rat DN model. The colocalization of P2X7R (a, red spots) or NLRP3 (b, red spots) and synaptopodin (a podocyte marker; green spots indicate alone localization and yellow spots indicate colocalization) in the frozen renal tissue section of the rat DN model (immunofluorescence microscopy ×400).

Article Snippet: Rabbit anti-P2X7R pAb (Alomone) , Ditto.

Techniques: Double Immunofluorescence Staining, Marker, Immunofluorescence, Microscopy

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Effects of ACOS on the high-glucose-induced expression of P2X7R/NLRP3 inflammasome in cultured podocytes. Podocytes were incubated in medium and medium containing 30 mM glucose and/or 50 μ g/mL ACOS, respectively. (a) After 6 h of incubation, cells were harvested. Then, the total RNA was extracted, and the relative mRNA expression levels of P2X7R, NLRP3, ASC, and procaspase-1 were measured by real-time quantitative PCR. (b) After 24 h of incubation, cells were lysed and the total lysates were used to determine the protein expression levels of P2X7R, NLRP3, ASC, active caspase-1 subunit P10, and β -actin by the Western blot assay. The relative protein expression level was expressed as the target protein/ β -actin protein ratio. Values are represented as mean ± SD (n = 3). ∗ P < 0.05 and ∗∗ P < 0.01 vs. control group, # P < 0.05 and ## P < 0.01 vs. HG group.

Article Snippet: Rabbit anti-P2X7R pAb (Alomone) , Ditto.

Techniques: Expressing, Cell Culture, Incubation, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Primer sequences for PCR analysis in animal experiments.

Article Snippet: A rabbit anti-P2X7R polyclonal antibody (1 : 100 dilution, Alomone) or rabbit anti-NLRP3 polyclonal antibody (1 : 100 dilution, Novus) and mouse anti-synaptopodin monoclonal antibody (1 : 50 dilution, Santa Cruz) were used as primary antibodies.

Techniques: Sequencing

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Primer sequences for PCR analysis in cellular experiments.

Article Snippet: A rabbit anti-P2X7R polyclonal antibody (1 : 100 dilution, Alomone) or rabbit anti-NLRP3 polyclonal antibody (1 : 100 dilution, Novus) and mouse anti-synaptopodin monoclonal antibody (1 : 50 dilution, Santa Cruz) were used as primary antibodies.

Techniques: Sequencing

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Primary and secondary antibodies for Western blot assays.

Article Snippet: A rabbit anti-P2X7R polyclonal antibody (1 : 100 dilution, Alomone) or rabbit anti-NLRP3 polyclonal antibody (1 : 100 dilution, Novus) and mouse anti-synaptopodin monoclonal antibody (1 : 50 dilution, Santa Cruz) were used as primary antibodies.

Techniques: Western Blot

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Effects of ACOS on the expression of P2X7R/NLRP3 inflammasome in the rat DN model. (a) Total RNA was extracted from renal cortex tissue, and the relative mRNA expression levels of P2X7R, NLRP3, ASC, and procaspase-1 were measured by real-time quantitative PCR. (b) Renal cortex tissue was lysed, and total lysates were analyzed by the Western blot assay with antibodies against P2X7R, NLRP3, ASC, active caspase-1 subunit P10, and β -actin, respectively. The relative protein expression level was expressed as the target protein/ β -actin ratio. Values are represented as mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01 vs. control group, # P < 0.05 and ## P < 0.01 vs. DN model group.

Article Snippet: A rabbit anti-P2X7R polyclonal antibody (1 : 100 dilution, Alomone) or rabbit anti-NLRP3 polyclonal antibody (1 : 100 dilution, Novus) and mouse anti-synaptopodin monoclonal antibody (1 : 50 dilution, Santa Cruz) were used as primary antibodies.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Double immunofluorescence staining of the podocyte marker and P2X7R/NLRP3 in renal tissues of the rat DN model. The colocalization of P2X7R (a, red spots) or NLRP3 (b, red spots) and synaptopodin (a podocyte marker; green spots indicate alone localization and yellow spots indicate colocalization) in the frozen renal tissue section of the rat DN model (immunofluorescence microscopy ×400).

Article Snippet: A rabbit anti-P2X7R polyclonal antibody (1 : 100 dilution, Alomone) or rabbit anti-NLRP3 polyclonal antibody (1 : 100 dilution, Novus) and mouse anti-synaptopodin monoclonal antibody (1 : 50 dilution, Santa Cruz) were used as primary antibodies.

Techniques: Double Immunofluorescence Staining, Marker, Immunofluorescence, Microscopy

Journal: Journal of Diabetes Research

Article Title: Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation

doi: 10.1155/2018/1390418

Figure Lengend Snippet: Effects of ACOS on the high-glucose-induced expression of P2X7R/NLRP3 inflammasome in cultured podocytes. Podocytes were incubated in medium and medium containing 30 mM glucose and/or 50 μ g/mL ACOS, respectively. (a) After 6 h of incubation, cells were harvested. Then, the total RNA was extracted, and the relative mRNA expression levels of P2X7R, NLRP3, ASC, and procaspase-1 were measured by real-time quantitative PCR. (b) After 24 h of incubation, cells were lysed and the total lysates were used to determine the protein expression levels of P2X7R, NLRP3, ASC, active caspase-1 subunit P10, and β -actin by the Western blot assay. The relative protein expression level was expressed as the target protein/ β -actin protein ratio. Values are represented as mean ± SD (n = 3). ∗ P < 0.05 and ∗∗ P < 0.01 vs. control group, # P < 0.05 and ## P < 0.01 vs. HG group.

Article Snippet: A rabbit anti-P2X7R polyclonal antibody (1 : 100 dilution, Alomone) or rabbit anti-NLRP3 polyclonal antibody (1 : 100 dilution, Novus) and mouse anti-synaptopodin monoclonal antibody (1 : 50 dilution, Santa Cruz) were used as primary antibodies.

Techniques: Expressing, Cell Culture, Incubation, Real-time Polymerase Chain Reaction, Western Blot