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Molecular Biology Tools> | Candidate shRNAs were designed using publicly available web tools (Invit- rogen Block-it and Genscript)....4), the membranes were cut into sections containing different target proteins and incubated with primary antibodies against Cav␣2␦1 (mouse, 1:1000, catalog #D219, Sigma), TSP4 (rabbit, 1:1000, custom made and validated against purified TSP4 pro- teins, Genscript, Piscataway, NJ), -actin (mouse, 1:10,000, cat- alog #MAB8929, validated against various -actin expressing cell lines, Novus Biologicals, LLC, Littleton, CO) overnight at 4 °C followed by horseradish peroxidase-conjugated secondary antibody (1:2000, Cell Signaling, Danvers, MA) for 1 h atroom temperature. | Get A Quote |
Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/... More