affinity purified rabbit anti p2x7r antibodies  (Alomone Labs)

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: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Oligonucleotide Primer List.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Antibody List.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Binding Assay, Transduction

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: To assess whether P2X7-R splice variants other than the full length, variant 1 are expressed in the WT and P2X7-KO mouse retina and also whether read through of variant 1 C-terminal mRNA occurs in the P2X7-KO mouse retina, PCR for (A) the central region of the P2X7-R which is common to variants 1, 2 and 3; (B) the C-terminus from within the start of the disruption in the P2X7-KO to the end of the mRNA (variant 1 only, primer pair A); and (C) the C-terminus from after the insertion deletion in the P2X7-KO mouse to the end of the receptor (variant 1 only, primer pair B) was completed. P2X7-R variant 1 C-terminal mRNA was absent (B–C) in the P2X7-KO mouse but N-terminal mRNA variants (likely variant 2 or 3) were still expressed (A). MW, molecular weight marker and the bright bar is 300 bp. (D–E) Protein analysis of P2X7-R splice variant expression was completed using Western blots of mouse retina (D) and cortex (E). An N-terminal specific P2X7-R antibody detected the protein transcript of variant 1 mRNA in WT samples 1 and 2 but not P2X7-KO samples 3 and 4 (68.41 kDa) of both retina and cortex. Protein for variant 2/3 (50.69 kDa/49.38 kDa) and 4 (17.2 kDa) was expressed in both WT and P2X7-KO retina and cortex. A monoclonal antibody against GAPDH was applied to the same blots and indicates that similar amounts of protein were loaded for WT and P2X7-KO samples.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Variant Assay, Molecular Weight, Marker, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Mice were dark adapted overnight and mixed (rod and cone) ERG responses were recorded in response to full field flashes from intensity −4.2 to 2.1 log cd.s/m 2 . (A–B) Representative mixed ERG waveforms for (A) WT and (B) P2X7-KO mice are presented. (C) There was no change in the amplitude of the rod photoreceptor derived component, a-wave, as a function of intensity. (D) The amplitude of the post-photoreceptor response, the b-wave was larger in P2X7-KO mice than in WT mice across almost all intensities tested. * indicates p<0.05, a significant difference between WT and P2X7-KO.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) Representative waveforms of the rod ERG recorded from WT (black) and P2X7-KO mice (grey) in response to a 2.1 log cd.s/m 2 intensity flash. The photoreceptor derived, rod PIII (modelled a-wave) was not altered in either (B) amplitude or (C) sensitivity in the P2X7-KO mouse when compared with the WT response. The post-photoreceptor, rod PII (modelled b-wave) was found to be (D) larger in amplitude and (E) faster to reach a maximum response. (F) The amplitude of the third of the inner retinal derived oscillatory potentials was significantly enhanced in the P2X7-KO mouse. * indicates p<0.05, a significant difference between WT and P2X7-KO.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) Representative waveforms of the cone ERG recorded from WT (black) and P2X7-KO mice (grey) in response to a 2.1 log cd.s/m 2 intensity flash. The post-photoreceptor, cone PII (modelled b-wave) was found to be larger in (B) amplitude but not faster to reach a maximum response (C) in the P2X7-KO mouse when compared with the WT response. (D) The photopic visual acuity, assessed using the optokinetic reflex to determine the spatial frequency threshold, was higher in the P2X7-KO mouse than in the WT. * indicates p<0.05, a significant difference between WT and P2X7-KO.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Transverse sections of paraffin embedded retina from (A) WT and (B) P2X7-KO mice were stained with hematoxylin and eosin for comparison. The gross retinal morphology was similar in both mice. Scale bar, 20 microns.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Staining

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) Immunohistochemistry on a transverse retinal section from a WT mouse showing P2X7-R immunoreactivity in the outer plexiform layer (OPL), inner nuclear layer (INL), and ganglion cell layer (GCL). (C) The retina does not label with the P2X7-R antibody following incubation with P2X7-R specific peptide. Scale bar, 20 microns.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques: Immunohistochemistry, Incubation

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) P2X7-R IR (green) colocalises with horizontal cells labelled with Calbindin (red) and their terminals. (B) P2X7-R IR (green) colocalises with rod bipolar cells labelled with PKC (red). (C) Inset of rod bipolar cell terminals. (D) P2X7-R IR (green) puncta are discretely expressed within photoreceptor terminals labelled with PSD-95 (red). (E) Cone bipolar cells (Type 2 and 6) labelled with a ZNP-1 antibody (green) colocalise with P2X7-R IR (red) including both the cone bipolar cell dendrites and terminals (F). P2X7-R IR (green) colocalises with amacrine cells labelled with calretinin (G) and in particular GABAergic amacrine cells (H). (I) Ganglion cells labelled with an antibody against GFP in the Thy1-HYFP mouse (pseudo-coloured red) were P2X7-R IR (pseudo-coloured green). Scale bar, 10 microns.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A) P2X7-R IR (red) is associated with but does not colocalise with ribbon synapses (ribeye, green) on bipolar cell terminals (VGLUT, blue). (B) Magnified inset from A, arrows indicate ribbon synapses associated with P2X7-R IR puncta. (C) P2X7-R IR (red) colocalises with conventional synapses (bassoon, green) distinct from bipolar cell terminals (VGLUT, blue). (D) Magnified inset from C, arrows indicate conventional synapses that colocalise with P2X7-R IR puncta. Co-localisation analysis indicates that P2X7-R IR puncta do not co-localise with ribbon synapses (E) but do show significant colocalisation with conventional synapses (F) in the IPL. Scale bars are 5 microns for A & C and 2 microns for B & D.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: Ribbons are indicated by arrows and labelled amacine cells are indicated by astericks. (A), (B) & (C) P2X7-R immunoreactivity labels amacrine cells adjacent to rod bipolar cell ribbon synapses in the IPL. (D) A conventional amacrine cell synapse is labelled for P2X7 immunoreactivity in close proximity to a rod bipolar cell terminal. Scale bars are 200 nm for A & B and 500 nm for C & D.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: PLoS ONE

Article Title: Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse

doi: 10.1371/journal.pone.0029990

Figure Lengend Snippet: (A–B) Müller cells, labelled with an antibody against glutamine synthetise (red) were found to be closely associated with P2X7-R-IR puncta in the IPL (green), but not co-localised in the IPL. (C) Astrocytes, labelled with glial fibrilliary acidic protein (GFAP; red) and P2X7-R-IR (green) were found to co-localise in retinal flat mount. (D) Microglia labelled with mouse anti-GFP (pseudo-coloured red) from CX3CR1-GFP heterozygous mice were also found to co-localise with P2X7-R-IR (pseudo-coloured green). Scale bar, 10 microns.

Article Snippet: The extracellular, N-terminal P2X7-R antibody directed against amino acid residues 136–152 of the receptor (Alomone labs, Cat. no#APR008) that was used for western blotting was also found to label similarly in both WT ( ) and P2X7-KO mice (data not shown).

Techniques:

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Briefly, PBMCs (1 × 10 6 ) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C.

Techniques:

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p < 0.0001 and * p < 0.05. Circulating monocytes purified from HD were incubated with inflammatory lipopolysaccharide (250 ng/ml) (C) or 2′3′- O -(4-benzoyl-benzoyl)adenosine5′-triphosphate (250 µM) (D) for 4 and 24 h. Flow cytometry analysis and representative plots of P2X7R expression are shown (C,D) , together with cumulative data from three independent experiments are presented (E) . Statistical significance was calculated by ANOVA-Student’s t-test * p < 0.05, ** p < 0.01.

Article Snippet: Briefly, PBMCs (1 × 10 6 ) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C.

Techniques: In Vitro, Quantitative RT-PCR, Isolation, Flow Cytometry, Purification, Incubation, Expressing

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.

Article Snippet: Briefly, PBMCs (1 × 10 6 ) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C.

Techniques: Immunohistochemistry, Immunofluorescence, Marker, Staining

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).

Article Snippet: Briefly, PBMCs (1 × 10 6 ) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C.

Techniques: Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .

Article Snippet: Briefly, PBMCs (1 × 10 6 ) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C.

Techniques: Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.

Article Snippet: Briefly, PBMCs (1 × 10 6 ) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C.

Techniques: Expressing, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p < 0.05, ** p < 0.01. Results are shown as mean ± SEM.

Article Snippet: Briefly, PBMCs (1 × 10 6 ) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .

Article Snippet: Briefly, PBMCs (1 × 10 6 ) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C.

Techniques: Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.

Article Snippet: Briefly, PBMCs (1 × 10 6 ) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C.

Techniques: Expressing, Activation Assay

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

Techniques:

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p < 0.0001 and * p < 0.05. Circulating monocytes purified from HD were incubated with inflammatory lipopolysaccharide (250 ng/ml) (C) or 2′3′- O -(4-benzoyl-benzoyl)adenosine5′-triphosphate (250 µM) (D) for 4 and 24 h. Flow cytometry analysis and representative plots of P2X7R expression are shown (C,D) , together with cumulative data from three independent experiments are presented (E) . Statistical significance was calculated by ANOVA-Student’s t-test * p < 0.05, ** p < 0.01.

Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

Techniques: In Vitro, Quantitative RT-PCR, Isolation, Flow Cytometry, Purification, Incubation, Expressing

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.

Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

Techniques: Immunohistochemistry, Immunofluorescence, Marker, Staining

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).

Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

Techniques: Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .

Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

Techniques: Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.

Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

Techniques: Expressing, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p < 0.05, ** p < 0.01. Results are shown as mean ± SEM.

Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .

Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

Techniques: Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

doi: 10.3389/fimmu.2017.01529

Figure Lengend Snippet: Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.

Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

Techniques: Expressing, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization *

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: P2X7 receptor-mediated dye uptake is enhanced by depletion of plasma membrane cholesterol and reduced by loading of cholesterol. A , example confocal images captured during 245-s time series to measure dye uptake. Time of image capture, in seconds, is shown. Ethidium bromide was added at 20 s, and 300 μ m BzATP was added at 45 s. The increase in fluorescence as a result of ethidium uptake ( red ) into P2X7-expressing cells ( green ) is apparent. B , human P2X7-mediated dye uptake was slower than mouse P2X7-mediated dye uptake ( n = 3 coverslips, ∼10–30 cells per coverslip, p < 0.001), normalized to human P2X7. C , measurements of cholesterol ( chol ) in TSA 201 cells. A 15-min preincubation with 5 or 15 m m MCD reduced cholesterol, 5 m m αCD for 15 min had no effect, and 100 μg/ml water soluble cholesterol for 30 min increased cholesterol ∼2-fold ( n = 3, *, p < 0.01, **, p < 0.001; ns , not significant). D , time course of human P2X7-mediated dye uptake, normalized to control. Uptake was enhanced by 5 m m MCD and inhibited by 100 μg/ml cholesterol ( n = 3–4, p < 0.05). E , 5 m m αCD has no effect on human P2X7-mediated dye uptake, normalized to the untreated condition ( n = 3, p > 0.05). F , rate of P2X7-mediated dye uptake 10–50 s after agonist application, normalized to control. Human P2X7 dye uptake was increased ∼5-fold by 5 m m MCD and almost abolished by 100 μg/ml cholesterol ( n = 66 alone, n = 64 MCD and n = 6 cholesterol, **, p < 0.001). Mouse P2X7 dye uptake was increased ∼2.2-fold by MCD and reduced by cholesterol ( n = 88 alone, n = 50 MCD and n = 4 cholesterol, **, p < 0.001). G , concentration-response curves for mouse P2X7 receptor-mediated dye uptake, with and without pretreatment with 5 m m MCD. Responses were normalized to the rate of increase in fluorescence in untreated cells stimulated with 300 μ m BzATP. H , time course of dye uptake mediated by the more active mouse P2X7k variant, stimulated with 30 μ m BzATP, normalized to the untreated condition ( n = 3, p < 0.05 for MCD, p < 0.001 for cholesterol). I , rat P2X7 dye uptake was enhanced by 5 m m MCD, but not significantly ( p = 0.07), and inhibited by 100 μg/ml cholesterol ( n = 3, p < 0.05), normalized to the untreated condition. J , in mouse bone marrow-derived macrophages, dye uptake via endogenous P2X7 was potentiated by 5 m m MCD and reduced by 100 μg/ml cholesterol ( n = 4, p < 0.001 for MCD, p < 0.01 for cholesterol), normalized to the untreated condition.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Fluorescence, Expressing, Concentration Assay, Variant Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization *

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: The enhancement in P2X7 receptor-mediated dye uptake by MCD treatment is independent of pannexin-1 and cytoskeletal changes. A , neither carbenoxolone (+ carb ; 30 μ m ) nor pretreatment with the caspase inhibitor zVAD-FMK (+ zVAD ; 5 μ m for 1 h) reduced human P2X7-mediated ethidium uptake following MCD treatment. ( n = 6, p > 0.05). B , preincubation with (−)-blebbistatin (+ bleb ; 100 μ m for 1 h), an inhibitor of non-muscle myosin 2, had no effect on the rate of mouse P2X7-mediated dye uptake, with or without MCD pretreatment ( n = 12, p > 0.05). C , the actin-stabilizing agent jasplakinolide (+ jasp ; 30 n m for 1 h) had no effect on human P2X7-mediated dye uptake with or without MCD treatment ( n = 4–8, p > 0.05). D , latrunculin-A (+ lat ; 5 μ m for 30 min), an inhibitor of F-actin polymerization, had no effect on the initial rate of mouse P2X7-mediated dye uptake, with or without MCD treatment ( n = 3–6, p > 0.05).

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization *

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: Acute changes in cholesterol affect P2X7 whole-cell current amplitude, sensitization and NMDG permeability. A , example traces of whole-cell patch clamp recordings from HEK 293 cells expressing the mouse P2X7 receptor and enhanced GFP, stimulated with repeated 5-s applications of 300 μ m BzATP followed by a 1-min wash. chol , cholesterol. B , mean current densities after one or more applications of BzATP, in untreated cells or cells pretreated with 5 m m αCD, 5 m m MCD, or 100 μg/ml cholesterol ( n = 3–18 cells, *, p < 0.01, **, p < 0.001; ns , not significant). pF , picofarads. C , example traces of whole-cell recordings from cells expressing the human P2X7 receptor, stimulated with repeated 5-s applications of 300 μ m BzATP followed by a 1-min wash. D , mean current density upon first agonist application, with and without 5 m m MCD pretreatment ( n = 18 cells, **, p < 0.001). E , -fold change in current density from first agonist application to last application (>10), where each point represents one cell. The solid line shows mean -fold change, and the dotted line is at 1. F–H , mouse P2X7 receptor currents in the NMDG + extracellular solution, stimulated with 300 μ m BzATP for 40 s. Mean peak current densities and the 10–90% rise time are shown ( n = 7–9, *, p < 0.01). I , TSA 201 cells expressing mouse P2X7 or human P2X7 receptors were untreated or treated with 5 m m MCD for 15 min and then surface-labeled with sulfo-NHS-LC-biotin. For mouse P2X7, 3 wells of a 6-well plate of cells were then solubilized in 350 μl of solubilization buffer, whereas for human P2X7, a T75 flask of cells was solubilized in 1 ml. The Western blot shows the total lysate ( T ) and surface-biotinylated sample ( B ) blotted for P2X7 and β-actin. MCD treatment did not affect the proportion of P2X7 receptors at the surface.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Permeability, Patch Clamp, Expressing, Labeling, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization *

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: The N terminus is important for the sensitivity of P2X7 receptor activity to cholesterol depletion. A , amino acid sequence alignment of the N-terminal region of human P2X1, P2X2, and P2X7 receptors, with conserved amino acids highlighted in gray , P2X1 aa20–23 and equivalent residues in P2X2 underlined , and the P2X7 residues Lys 17 and Val 18 in red. B , time course of dye uptake mediated by the K17R/V18M mutant and wild type P2X7, alone, pretreated with MCD and loaded with cholesterol ( chol ), normalized to the untreated WT P2X7. C , bar graph of the relative rate of dye uptake normalized to the untreated WT P2X7. Dye uptake mediated by the K17R/V18M mutant is not potentiated by MCD, but is inhibited by cholesterol loading (WT: n = 14 alone, n = 11 MCD, n = 3 cholesterol; mutants: n = 12 alone, n = 11 MCD, n = 3 cholesterol; *, p < 0.05, **, p < 0.001; ns , not significant). D , whole-cell patch clamp recordings of human P2X7 K17R/V18M receptor ± 5 m m MCD pretreatment, stimulated with repeated 5-s applications of 300 μ m BzATP followed by a 1-min wash. E , mean peak current density upon first application of BzATP. MCD pretreatment potentiates initial current density ( n = 10–17 cells, **, p < 0.001), but the extent of potentiation is less than the wild type receptor ( p < 0.05, two-way analysis of variance). pF , picofarads.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Activity Assay, Sequencing, Mutagenesis, Relative Rate, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization *

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: Cholesterol-sensing within the proximal C terminus of the P2X7 receptor. A , amino acid sequence of the human P2X7 N terminus and TMD1, and the end of the TMD2 and proximal C terminus. The CRAC motifs are underlined , with constituent residues in bold and central tyrosines in red. Arrows indicate the amino acids reported to interact with phosphatidylinositol 4,5-bisphosphate , and the cluster of hydrophobic residues downstream of these is in bold/blue . The 18-amino acid cysteine-rich region is highlighted in gray , and its cysteines are in bold/green. B , bar graph of the relative rates of dye uptake ± 5 m m MCD for P2X7 Tyr to Phe mutants normalized to the untreated WT P2X7 (WT: alone n = 16, MCD n = 5; mutants: n = 3–6; *, p < 0.01, **, p < 0.001; ns , not significant). C , cells expressing P2X7 WT or P2X7 mutants were surface-labeled with sulfo-NHS-LC-biotin. The Western blot shows the total lysate ( T ) and surface-biotinylated sample ( B ) blotted for P2X7 and β-actin. The Y51F mutation inhibited surface and total expression of the receptor. D , bar graph of the relative rates of dye uptake ± 5 m m MCD, for hydrophobic to alanine mutants normalized to the untreated WT P2X7 (WT: n = 9; mutants: n = 3–6; *, p < 0.05, **, p < 0.001). E , cells expressing P2X7 WT or P2X7 mutants were surface-labeled with sulfo-NHS-LC-biotin. The Western blot shows the total lysate ( T ) and surface-biotinylated sample ( B ) blotted for P2X7 and β-actin. Surface expression was inhibited by V401A, F403A, and V404A mutations.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Sequencing, Expressing, Labeling, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization *

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: Bromopalmitate pretreatment inhibits surface expression of the P2X7 receptor but does not abolish sensitivity to MCD. A and B , time course of dye uptake by mouse P2X7 receptor ( A ) and human P2X7 receptor ( B ) after 16 h of pretreatment with 100 μ m BrP or DMSO (control). P2X7 receptor-mediated dye uptake in BrP-treated cells was significantly potentiated by 5 m m MCD ( n = 4 for mouse, n = 3 for human, p < 0.05). C , HeLa cells expressing mouse P2X7-FLAG receptors were immunostained by live labeling with anti-FLAG antibody. BrP reduced the surface expression of mouse P2X7-FLAG, but MCD treatment did not ( scale bar = 20 μm, n = 32–40 cells per condition, **, p < 0.001; ns , not significant). D , Western blot of TSA 201 cells expressing mouse P2X7 shows that pretreatment with BrP reduces total expression of the receptor.

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Expressing, Labeling, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization *

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: The cysteine-rich region within the proximal C terminus of P2X7 is important for normal pore dilation. A , time course of dye uptake mediated by P2X7 Δ18aa ± 5 m m MCD normalized to the untreated WT P2X7. B , relative rates of dye uptake mediated by P2X7 Δ18aa ± MCD, normalized to untreated WT P2X7 (WT: n = 6 alone, n = 2 MCD; mutants: n = 7 alone, n = 4 MCD; *, p < 0.05). C , Western blot shows that total ( T ) and surface-biotinylated ( B ) expression of P2X7 Δ18aa was similar to WT P2X7 ( panel B ). D , example whole-cell current recording for P2X7 Δ18aa stimulated with repeated 5 s applications of 300 μ m BzATP. Currents had slow activation kinetics, exhibited run down, and were flickery. E , the mean peak current density for the first agonist application appeared to be reduced by MCD treatment, although this was not significant (alone n = 13, MCD n = 8, p > 0.05). F , bar graph of the relative rates of dye uptake ± 5 m m MCD for P2X7 Cys to Ala mutants, normalized to untreated WT P2X7. (WT: alone n = 8, MCD n = 11; Cys to Ala mutants: n = 3–6; *, p < 0.05, **, p < 0.001; ns , not significant). G , Western blot shows that surface expression of C363A and C373A mutants was similar to WT, whereas C371A and C377A mutants had reduced surface expression. H , whole-cell recording of P2X7 C363A receptor currents, stimulated with repeated 5-s applications of 300 μ m BzATP. I , P2X7 C363A mean peak current density upon first application of BzATP was potentiated by 5 m m MCD pretreatment ( n = 5, *, p < 0.05).

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Western Blot, Expressing, Activation Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: P2X7 Receptor Inhibition Interrupts the Progression of Seizures in Immature Rats and Reduces Hippocampal Damage

doi: 10.1111/cns.12272

Figure Lengend Snippet: P2X7R protein levels following neonatal status epilepticus (SE). (A) Representative Western blot (n = 1 per lane) and graph (n = 4 per group) showing P2X7R protein levels in the rat hippocampus at postnatal day 1, 5, 10, and 30. (B) Representative Western blot (n = 1 per lane) and graph (n = 4 per group) showing P2X7R protein levels increase following neonatal SE. (C) Immunohistochemistry (4 h) showing (top) P2X7R (green) staining within the stratum lucidum (mossy fiber terminals) and around cell bodies of NeuN‐positive cells and (bottom) P2X7R (red) with synaptophysin (syn, green). Scale bar, 50 μm. (D) Graph quantifying IL1β levels following neonatal SE (n = 5 per group). *P < 0.05 compared with control.

Article Snippet: For P2X7R immunohistochemistry, sections were permeabilized and blocked in goat serum followed by incubation with antibodies against P2X7R (1:200; Alomone Labs, Jerusalem, Israel), NeuN (1:500; Millipore Ireland B.V.), or synaptophysin (1:500; Sigma‐Aldrich) overnight at 4°C.

Techniques: Western Blot, Immunohistochemistry, Staining