Journal: Molecular Pain
Article Title: Involvement of NIPSNAP1, a neuropeptide nocistatin-interacting protein, in inflammatory pain
Figure Lengend Snippet: Analysis of NIPSNAP1 mRNA expression induced by PGE 2 . (a) RT-PCR and real-time PCR analysis of NIPSNAP1 expression induced by 5 µM PGE 2 in DRG cells. (b) RT-PCR analysis of the expression of mRNA encoding EP subtypes in DRG cells. White arrowhead shows EP3β. (c) Effect of EP agonists on the expression of NIPSNAP1 mRNA. DRG cells were incubated for 1 h in the presence of each EP agonist (1 µM), and subjected to RT-PCR and real-time PCR analysis. (d) Effect of the protein kinase A inhibitor H-89 on the PGE 2 -induced expression of NIPSNAP1 mRNA. DRG cells incubated with 5 µM PGE 2 for 1 h in the absence and presence of 20 µM H-89 were subjected to RT-PCR and real-time PCR analysis. The upper and lower panels show representative results of RT-PCR and real-time PCR analysis, respectively. Data are expressed as the mean ± SEM ( n = 3–4). **p
Article Snippet: Reverse-transcription polymerase chain reaction (RT-PCR) amplifications of NIPSNAP1 and EPs were performed as follows: 1 cycle at 94℃ for 1 min followed by 10 cycles at 94℃ for 1 min and at 72℃ for 3 min, 15 cycles at 94℃ for 1 min, 65℃ for 2 min, and at 72℃ for 30 s, and 10 cycles at 94℃ for 1 min, 62℃ for 2 min, and at 72℃ for 30 s, and 1 cycle at 72℃ for 2 min. RT-PCR amplification of GAPDH was performed as follows: 1 cycle at 94℃ for 1 min followed by 35 cycles at 94℃ for 1 min, 60℃ for 1.5 min, and at 72℃ for 30 s, and finally 1 cycle at 72℃ for 7 min. mRNA levels were measured using quantitative real-time PCR with a StepOne System (Applied Biosystem) and GoTaq qPCR master mix (Promega).
Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Incubation