rabbit anti extracellular p2x7r igg  (Alomone Labs)

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

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

Journal: Journal of Neuroinflammation

Article Title: Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

doi: 10.1186/s12974-020-02058-y

Figure Lengend Snippet: The effects of L-NAME and/or PDI knockdown on the amounts of SNO-thiol and total thiol-PDI or -P2X7R, and PDI-P2X7R bindings following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-PDI and PDI-P2X7R bindings without affecting PDI expression and the amount of total thiol-PDI under physiological condition. Thus, S -nitrosylation ratio of PDI is decreased. L-NAME abrogates SE-induced alterations in PDI-P2X7R bindings and S -nitrosylation ratio of PDI, except PDI expression. As compared to control siRNA (C), PDI siRNA (P) abolishes the changes in PDI-P2X7R bindings and S -nitrosylation ratio of P2X7R following SE, except its expression. a Representative Western blot for S -nitrosylation and thiolization on PDI and its expression. b Quantification of analyses of S -nitrosylation and thiolization on PDI and its expression. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p

Article Snippet: The primary antibodies were mouse anti-PDI (1:1,000, Abcam, ab2792) and rabbit anti-extracellular P2X7R IgG (1:200, Alomone labs, APR-008).

Techniques: Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

doi: 10.1186/s12974-020-02058-y

Figure Lengend Snippet: Scheme of the role of PDI-mediated S -nitrosylation of P2X7R in its trafficking to the cell membrane. SE elevates NO synthesis, which S -nitrosylates PDI. SNO-PDI transfers NO to the reduced (immature) P2X7R. In turn, SNO-P2X7R is oxidized by denitrosylases (presumably Trx-1 [ 8 ]), which facilitates its trafficking to the cell surface

Article Snippet: The primary antibodies were mouse anti-PDI (1:1,000, Abcam, ab2792) and rabbit anti-extracellular P2X7R IgG (1:200, Alomone labs, APR-008).

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

doi: 10.1186/s12974-020-02058-y

Figure Lengend Snippet: The effects of L-NAME and PDI knockdown on the surface expression of P2X7R. As compared to control animals, SE increases the membrane P2X7R expressions, which are abrogated by both L-NAME and PDI siRNA. a Representative Western blot for surface expression of P2X7R following SE. b Quantification of analyses of P2X7R expression in membrane fractions. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p

Article Snippet: The primary antibodies were mouse anti-PDI (1:1,000, Abcam, ab2792) and rabbit anti-extracellular P2X7R IgG (1:200, Alomone labs, APR-008).

Techniques: Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

doi: 10.1186/s12974-020-02058-y

Figure Lengend Snippet: The effects of L-NAME on the amounts of SNO-thiol- and total thiol-P2X7R following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-P2X7R and increased that of total thiol-P2X7R without altering P2X7R expression under physiological condition. Thus, S -nitrosylation ratio of P2X7R is decreased. SE upregulates P2X7R expression and S -nitrosylation ratio of P2X7R due to the reduced the amount of total thiol without the altered that of SNO-thiol-P2X7R. L-NAME inhibits these alterations induced by SE, except P2X7R expression. a Representative Western blot for S -nitrosylation and thiolization on P2X7R and its expression. b-e Quantification of analyses of S -nitrosylation and thiolization on P2X7R and its expression. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p

Article Snippet: The primary antibodies were mouse anti-PDI (1:1,000, Abcam, ab2792) and rabbit anti-extracellular P2X7R IgG (1:200, Alomone labs, APR-008).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Bacterial lipopolysaccharide and antimicrobial LL-37 enhance ICAM-1 expression and NF-κB p65 phosphorylation in senescent endothelial cells

doi: 10.3892/ijmm.2019.4294

Figure Lengend Snippet: Role of FPR2 and P2X7 receptors in senescent and non-senescent endothelial cells. (A) Non-senescent HUVECs were preincubated with WRW4 (0.1 or 1 µ M) or KN-62 (0.1 or 1 µ M) for 30 min, and then incubated with LL-37 (5 µ g/ml) for 24 h. Alternatively, non-senescent HUVECs were preincubated with a combination of WRW4 (0.1 µ M) and KN-62 (0.1 µ M) for 30 min, and then incubated with LL-37 (5 µ g/ml) for 24 h. ICAM-1 protein expression levels were analyzed by western blotting. Relative expression of ICAM-1/GAPDH was calculated as a ratio to LL-37-stimulated cells without antagonists. Data are presented as the mean ± SD of at least three independent experiments. (B) Cell surface expression levels of LL-37 receptors FPR2 and (C) P2X7 were analyzed in senescent and non-senescent HUVECs by flow cytometry. Relative expression in senescent cells was calculated as a ratio to non-senescent cells. Data are presented as the mean ± SD of at least four independent experiments. * P

Article Snippet: Polyclonal anti-P2X7 (cat. no. APR-008; Alomone Labs), normal rabbit IgG (cat. no. PM035; Medical and Biological Laboratories, Ltd.) and PE-conjugated donkey anti-rabbit IgG (cat. no. 406421; BioLegend, Inc.) were also used for flow cytometry.

Techniques: Incubation, Expressing, Western Blot, Flow Cytometry

Journal: The Journal of Neuroscience

Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

doi: 10.1523/JNEUROSCI.0852-17.2017

Figure Lengend Snippet: Spinal P2X7Rs are critically involved in the development of morphine tolerance. A – D , Effects of intrathecal injections of A740003 (0.1 nmol), a selective P2X7R antagonist, on the development of morphine tolerance. A , Thermal and ( B ) mechanical nociceptive threshold in CTR- ( n = 7), MS- ( n = 7), MS/A740003- ( n = 7), and A740003- ( n = 6) treated rats. Latency to withdraw from stimulus, tail-flick latency (TFL) and paw withdrawal thresholds (PWTs), are reported as a percentage of the maximum possible effect (MPE). **** p

Article Snippet: Free-floating spinal cord sections were incubated overnight at 4°C in mouse α-CD11b antibody (1:150; CBL1512 EMD, Millipore), rabbit α-P2X7R antibody (1:150; APR-008, Alomone Labs), rabbit α-μ-receptor antibody (1:500; AOR-011, Alomone Labs), rabbit α-Ki67 antibody (1:500; ab16667, Abcam), rabbit α-Iba1 antibody (1:1000; 019-19741, Wako).

Techniques: Tail Flick Test