Fig 1.
Immunofluorescence of SNAP-25, SV2A and TRPV1.
1A–1C: SNAP-25-positive neurons were observed in one-week-old DRG cultures, and positive staining was located both in the periphery of the cytoplasm and neuronal processes. The staining pattern did not change except that the number of SNAP-25-positive cells increased with longer culture periods. 1D–1F: Compared with the IF staining of SNAP-25, the positive staining for SV2A was mainly distributed in the cytoplasm and was less highly expressed along neuronal processes until 3 weeks of culture. Moreover, weak and scattered IF positivity for SV2A was observed in nuclei in the cells with shorter culture periods. 1G–1I: Much like SNAP-25, TRPV1 was also expressed in cultured DRG neurons after 1 week of culture. Initially, the positivity mainly appeared around the surface of the cell body. In the 2- and 3-week cultures, the positive immunofluorescence became much stronger. Obvious immunoreactivity for TRPV1 was observed in both the cytoplasm and processes when cells were cultured for up to 3 weeks, especially along the neuronal processes, as shown in the inset in 1I (bar = 50 μm).
Fig 2.
Expression of SNAP-25, SV2A and TRPV1 by SDS-PAGE and western blot.
2A: The distribution of SNAP-25 IF-positive neurons as a percentage of DAPI positive cells was dramatically increased after 2 weeks of culture. 2B: The percentage of TRPV1-positive neurons showed a similar pattern to that observed for SNAP-25. 2C: WB assays show that SNAP-25, SV2A and TRPV1 are strongly expressed in the extracts from 3-week-old DRG cultures, which confirms that 3-week-old cultured DRG neurons are a good cell model for studying BoNT/A action. 20 μg protein/lane; tubulin was used as a loading control.
Fig 3.
Co-immunoprecipitation and WB of TRPV1 and BoNT/A.
After 1 hour of 1nmol of BoNT/A treatment in 3-week-old DRG neuron cultures, the membrane protein extracts were incubated with either anti-BoNT/A antibody or anti-TRPV1 antibody overnight at 4°C and the precipitated proteins of interested were probed by western-blot using the cognate antibody (Top and Middle panels). Tubulin was used as a loading control (Lower panel).
Fig 4.
Immunofluorescent colocalization of TRPV1 with BoNT/A or cleaved SNAP-25.
Top-panel: After cultured DRG neurons were exposed to BoNT/A for 60 min, BoNT/A immunoreactivity was observed to colocalize with TRPV1 in the membranes of both soma and neurites (inset). Middle-panel: BoNT/A IF reactivity moved into the cytoplasm when the exposure time was increased to 24 hours, and the double labeling of BoNT/A and TRPV1 appeared partially co-localized; meanwhile, the IF double labeling was not easily observed along neuronal processes in these cells (inset). Lower-panel: Under microscope, cleaved SNAP-25 was observed to be distributed in a punctate pattern along the cell membrane and was colocalized (dotty) with TRPV1 after DRG neurons were treated with BoNT/A for 24 hours (bar = 50 μm).
Fig 5.
Functional interaction between BoNT/A and TRPV1 in cultured DRG neurons.
5A, 5B. The influence of anti-TRPV1 antibody applied before BoNT/A exposure on the percentage of SNAP-25 cleaved by BoNT/A. 5C, 5D. The effects of different exposure times of BoNT/A on the expression of TRPV1 in DRG neuron membrane extracts, as analyzed by WB.