anti rat p2x3 receptor  (Alomone Labs)

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: Daily i.t. injection of the selective P2X3 receptor antagonist A317491 attenuates morphine‐induced anti‐nociceptive tolerance. (A) Schematic of the experimental timeline. (B) Thermal and (C) mechanical thresholds measured daily in all groups (n = 6; *P < 0.05 vs. saline group; # P < 0.05 vs. morphine group).

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Injection

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: (A) Expression of P2X3 receptor and (B) number of P2X3 receptor‐positive cells per section per animal in DRGs increase after repeated morphine treatment. The DRGs were removed 2 h after morphine injection on day 7 (n = 6,*P < 0.05 vs. saline (S) group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Expressing, Injection

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: Morphine‐induced P2X3 receptor up‐regulation is co‐localized with IB4 and peripherin in the DRG. (A) Confocal images of P2X3 receptor (green) and ionized Ca2+‐binding adapter molecule 1 (Iba1) (red) immunofluorescence with no co‐localization. (B) Immunofluorescence images of P2X3 receptor (red) and IB4 (green) co‐localization in DRGs. (C) There was less co‐localization of P2X3 receptor (green) with NF200 (red) in myelinated neurons. (D) Co‐localization of P2X3 receptor (green) with peripherin (red) in unmyelinated/small‐calibre neurons. DRG samples from the 7 day morphine group (scale bars: A/B, 80 μm; C/D, 20 μm).

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Binding Assay, Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: Involvement of PKCε in morphine (Mor)‐induced antinociceptive tolerance and P2X3 receptor expression. (A) The phosphorylation of PKCε in the DRG increased time dependently with chronic Mor treatment. (B) The inhibitor of PKCε, ε‐V1‐2, reversed the increased P2X3 receptor expression induced by repeated Mor treatment. (C) PKCε co‐localized with P2X3 receptor in DRGs (scale bar: 50 μm for the up; 20 μm for the down). (D) Mor tolerance was attenuated when Mor was administered with ε‐V1‐2 for 7 days (n = 6,*P < 0.05 vs. saline (S) group; # P < 0.05 vs. morphine group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Expressing, Injection

Journal: British Journal of Pharmacology

Article Title: Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

doi: 10.1111/bph.14191

Figure Lengend Snippet: Epac is responsible for the chronic morphine (Mor)‐induced phosphorylation of PKCε and increase in P2X3 receptor expression. (A) The expression of Epac in the DRG increased time dependently after chronic Mor exposure. (B) The inhibitor of Epac, ESI‐09, reversed the increased P2X3 receptor expression and phosphorylation of PKCε induced by repeated morphine treatment. (C) Epac co‐localized with P2X3 receptor in DRGs (scale bar: 50 μm for the left; 20 μm for the right). (D) Morphine tolerance was attenuated by co‐administration of morphine and ESI‐09 for 7 days (n = 6,*P < 0.05 vs. saline (S) group; # P < 0.05 vs. morphine group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology).

Techniques: Expressing, Injection

Journal: Acta neuropathologica communications

Article Title: Adenosine triphosphate drives head and neck cancer pain through P2X2/3 heterotrimers.

doi: 10.1186/2051-5960-2-62

Figure Lengend Snippet: Figure 1 ATP, P2X2, and P2X3 in human HNSCC microenvironment and pain. a. Representative HPLC chromatograms showing ATP peaks from tumor and matched normal tissue harvested from the same patient. b. Tumor tissues (Ipsi) had higher levels of ATP compared to matched normal sites (Contra) (n = 10, Student’s t-test). c. Pain scores of functional sharpness and intensity were significantly higher than spontaneous sharpness and intensity, respectively (n = 13, Student’s t-test). d. Mean scores of functional pain (Q2, 4, 6, 7, 8) were significantly higher than spontaneous pain (Q1, 3, 5) (Student’s t-test). e. ATP concentration in extracted cancer tissue correlated positively with mean pain scores (linear regression). f. Representative H&E and immunofluorescence staining (P2X2, P2X3, merged) of a human tongue SCC. Sections were taken from adjacent sections of the SCC. Scale bar: 100 μm.

Article Snippet: Control experiments were performed by incubation in secondary antibody alone and by applying P2X2 blocking peptides (Santa Cruz Biotechnology), and P2X3 blocking peptides (Alomone Labs).

Techniques: Functional Assay, Concentration Assay, Immunofluorescence, Staining

Journal: Acta neuropathologica communications

Article Title: Adenosine triphosphate drives head and neck cancer pain through P2X2/3 heterotrimers.

doi: 10.1186/2051-5960-2-62

Figure Lengend Snippet: Figure 3 HNSCC induces neuronal P2X2/3 plasticity that is reversed by anti-NGF. a. Sustained ATP current (top panel) is enhanced and prolonged following SCC co-culture; anti-NGF added into the co-culture reduced the sustained ATP current. Transient ATP current (lower panel) is not affected by either co-culture or anti-NGF. b. Sustained ATP current density is increased by co-culture (One-way ANOVA), and is reversed by anti-NGF. c. Dot plot of sustained ATP current in different diameter neurons. Co-culture increased current in medium-sized TG neurons; this increased current was reversed by anti-NGF. d. Representative immunofluorescence images of P2X2 and P2X3 expression in TG neurons. e. HNSCC co-culture increased the percentage of neurons expressing P2X2 and P2X3 subunits. Anti-NGF treatment significantly reduced percentage of neurons expressing P2X3 but not P2X2 subunits. The significant increase in the percentage of neurons that express both subunits following co-culture was reversed by anti-NGF application (One-way ANOVA). f. P2X2 immunofluorescence intensity was not changed after co-culture or anti-NGF treatment. P2X3 immunofluorescence intensity was significantly increased following co-culture, and was reduced by anti-NGF (one-way ANOVA). g. In mice with tongue HNSCC, mRNA expression for P2X2 was increased, while P2X3 expression was unchanged in TG neurons (Student’s t-test).

Article Snippet: Control experiments were performed by incubation in secondary antibody alone and by applying P2X2 blocking peptides (Santa Cruz Biotechnology), and P2X3 blocking peptides (Alomone Labs).

Techniques: Co-Culture Assay, Immunofluorescence, Expressing