Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 2 Effect of UTP and suramin on IA of small-diameter and FG-labeled TG neurons, with diameters ranging from 18 to 39 μm in control rats. (A) Fluorescence microscopic view of TG neurons from control rats. (a) Retrograde labeling of TG neurons (blue) innervating whisker pad skin. (b) P2Y2 receptor-positive (green) TG neurons were seen in the section of TG. (c) The merged images (purple) of retrograde labeling of TG neurons and P2Y2 receptor-positive TG neurons from the same section, indicating co-localization. (d) Retrograde labeling of TG neurons (blue) innervating whisker pad skin in cultured TG neurons. (B) Electrophysiology recording for small-diameter and FG-labeled TG neurons in control rats. (a) Representative traces showing that the application of 30 μM UTP reduced IA. Suppression of the mean peak amplitudes of IA seen after UTP application was antagonized by suramin 100 μM. (b) Current–voltage relationship for the effects of UTP and suramin on IA. Each value represents the mean ± SEM (con: 0.14 ± 0.01 nA, n = 9; UTP: 0.09 ± 0.01 nA, n = 20, p < 0.05 vs control; suramin: 0.13 ± 0.01 nA, n = 9). IA was initiated via a prepulse (100 ms) of −120 mV and test pulses (400 ms) from −60 to +60 mV in a 10 mV step.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Labeling, Control, Fluorescence, Whisker Assay, Cell Culture

Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 4 Effect of UTP on the expression levels of IA subunits in control TG neurons. (A) Double-immunostaining revealed the expression of Kv1.4, Kv3.4, Kv4.2 and Kv4.3 subunits in P2Y2 receptor-positive neurons in the ION-CCI TG sections. The P2Y2 receptor-positive TG neurons also expressed Kv1.4, Kv3.4, Kv4.2 and Kv4.3, respectively, n = 5 rats. (B) Reduction in the mRNA levels of IA subunits by UTP in cultured TG neurons from control rats. Treatment with suramin (100 μM) in the UTP-incubated (30 μM) TG neurons for 16 h in control rats reversed the decrease in the mRNA levels of Kv1.4, Kv3.4, Kv4.2, and Kv4.3 subunits. n = 10 samples in each group, *, p < 0.05, **, p < 0.01 vs control; ##, p < 0.01 vs UTP.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Expressing, Control, Double Immunostaining, Cell Culture, Incubation

Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 5 The role of P2Y2 receptors in mechanical hyperalgesia in ION-CCI rats. (A) The peripheral target injection to TG of suramin (0.3- 3 μg/μl) reduced mechanical allodynia in the whisker pad. n = 6-8, **, p < 0.01 compared with injection of saline, ##, p < 0.01 compared with injection of high-dose suramin. Suramin led to a time- and dose-dependent increase in PWT, this anti-allodynia effect started 10 min after the suramin injection and remained for at least 45 min. (B) The peripheral target injection to TG of P2Y2 antisense oligodeoxynucleotides significantly alleviated mechanical allodynia of the whisker pad. n = 5, *, p < 0.05, **, p < 0.01 compared with injection of saline. The effect started at 6 h and persisted for at least 120 h. (C) Western blots showed successful suppression of P2Y2 receptor expression in TG by P2Y2 receptor antisense oligodeoxynucleotides treatment n = 4 for each group, **, p < 0.01.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Injection, Whisker Assay, Saline, Western Blot, Expressing

Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 6 Difference of IA channel expression in TG between sham and ION-CCI rats. (A) Double-immunostaining for P2Y2 receptors and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3 on TG neurons in sham and ION-CCI sections, respectively. (B) Percentages of numbers of Kv1.4, Kv3.4, Kv4.2 and Kv4.3 subunits in P2Y2 receptor-positive neurons are significantly decreased in TG from ION-CCI rats compared with sham rats. n = 4 rats, **, p < 0.01). (C) Changes in the mRNA levels of IA subunits in TG after P2Y2 receptor antisense oligodeoxynucleotides treatment. The mRNA levels of Kv1.4, Kv3.4 and Kv4.2 were significantly decreased in the saline group of ION-CCI rats compared with the sham rats. They were reversed after P2Y2 receptor antisense oligodeoxynucleotides treatment. n = 5-9 rats, *, p < 0.05, **p < 0.01 compared with saline groups. There was no difference in the levels of Kv4.3 mRNA among the groups. n = 6-8 rats, p > 0.05.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Expressing, Double Immunostaining, Saline

Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 7 Role of ERK pathway in activation of P2Y2 receptors mediates an inhibition of IA channels on small-diameter TG neurons in control rats. (A) Comparison of the phosphorylation of ERK1/2 in TG from sham and ION-CCI rats. Western blot results showed that the level of ERK1/2 phosphorylation was significantly increased in the ipsilateral TG after ION-CCI, compared with that from the sham group. n = 5 for each group *, p < 0.05. (B) In TG from ION-CCI rats, treatment with P2Y2 receptor antisense oligodeoxynucleotides (15 μg/50 μl) significantly decreased the expression of ERK protein in TG. n = 5 for each group, **, p < 0.01.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Activation Assay, Inhibition, Control, Comparison, Phospho-proteomics, Western Blot, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Intermediate Conductance Calcium‐Activated Potassium Channel Activation in Stem Cell Antigen‐1 Positive Cells Contributed to Vascular Restenosis

doi: 10.1161/JAHA.125.045943

Figure Lengend Snippet: A , Effect on migration. ATP increased Sca‐1 + cell migration, which was significantly inhibited by the IK Ca channel blocker TRAM34 ( a ). MCP‐1 also enhanced migration, and TRAM34 similarly suppressed this increase ( b ). (Statistical significance was confirmed by HC3‐robust interaction model with 6 biological replicates.) B , The ATP‐enhanced migration of Sca‐1 + cells was significantly abolished by IK Ca channel inhibition with shKCNN4 ( P =0.0360, HC3‐robust interaction model, n=5). C , The proliferation of Sca‐1 + cells induced by ATP ( a , b ) and PDGF‐BB ( c ) was significantly suppressed by inhibition of IK Ca channels with TRAM34 or shKCNN4 ( P values are indicated on the graph; HC3‐robust interaction model, n=5). ATP indicates adenosine triphosphate; CON, control; HC3, heteroskedasticity‐consistent robust SE; IK Ca , intermediate conductance calcium‐activated potassium channel; MCP‐1, monocyte chemoattractant protein‐1; PDGF‐BB, platelet‐derived growth factor‐BB; Sca‐1 + , stem cell antigen‐1 positive; shKCNN4, a short hairpin RNA construct specifically designed to target and silence the expression of the KCNN4 gene; and TRAM34, 1‐ [(2‐chlorophenyl) diphenylmethyl]‐1h‐pyrazole.

Article Snippet: They were then blocked with BSA and incubated overnight with an anti‐ KCNN4 receptor (extracellular) antibody (1:200, Alomone, Jerusalem, Israel, #APR‐102).

Techniques: Migration, Inhibition, Control, Derivative Assay, shRNA, Construct, Expressing