0.01; ***, 0.001 weighed against no Cav21; #, 0.05; ###, 0.001 between adjacent doses by one-way ANOVA with Bonferroni post-tests. and changes in synaptic transmission in mice overexpressing Cav21. Importantly, TSP4/Cav21-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cav21-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development. for 20 min at 4 C. The supernatant was incubated with anti-TSP4 polyclonal antibody (guinea pig, 1:750, validated previously; Ref. 36) overnight at 4 C. Protein A/G-agarose beads (Thermo, Waltham, MA) were then added, incubated for PIK-293 2 h at 4C, and washed with protein extraction buffer. The antibody-captured proteins were eluted in non-reducing condition with low pH elution buffer (Thermo, Waltham, MA) at room temperature, and the same volume of control supernatant or immunocomplex samples was analyzed by Western blots under non-reducing conditions. Solid-phase Binding Briefly, FLAG-Cav21cDNA was transiently transfected into the tsA-201 cell line stably expressing Cav2.2 and Cav3 (a gift from Dr. D. Lipscombe from Brown University (37) by Lipofectamine 2000 (Invitrogen). The transfected cells were PIK-293 washed and extracted in protein extraction buffer (50 mm Tris, 150 mm NaCl, 1 mm EDTA, 0.1% Triton-X, pH 7.4) in 2C3 days. The cell lysate was incubated on ice for 15 min, then centrifuged 13,000 at 4 C for 20 min. The supernatant was rotating-incubated with anti-FLAG M2 agarose affinity resin (Sigma) for 2 h at 4 C and washed with protein extraction buffer. FLAG-Cav21 was eluted in elution buffer (0.1 m glycine, pH 3.5) and stored at ?20 C until use. The reagents for solid-phase binding were from Invitrogen. Recombinant TSP4 proteins (80 g/ml) were immobilized onto a 96-well polystyrene plates (Thermo, Waltham, MA) overnight at 4 C in coating buffer A. All further incubations were carried out at room temperature for 1 h, and proteins or antibodies were diluted in assay buffer containing bovine serum albumin SHGC-10760 (BSA). After washing and blocking, the plates were incubated with FLAG-Cav21, washed, then incubated with mouse monoclonal anti-FLAG antibodies (1:1000; catalog #F1804, validated against FLAG fusion proteins by Sigma) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. The bound FLAG-Cav21 complexes were detected by measuring a color reaction at 450 nm after adding tetramethylbenzidine for 15 min followed by adding sulfuric acid to stop the reaction. Surface Plasmon Resonance Binding (38) All experiments were carried out using BIAcore 3000 and CM5 Sensor Chip (GE Healthcare) at 25 C. Cav21antibody (mouse, catalog #D219, Sigma) was coupled to the dextran matrix of a CM5 sensor chip using Amine Coupling kit as described (39). The antibody specificity for Cav21 PIK-293 was confirmed with tissue samples from Cav21knock-out mice (Fig. 4= 3) showing null Cav21 expression ( 0.05 compared with non-injury (test. 0.001 vehicle injected WT CKO mice by one-way ANOVA with Dunnett’s post hoc test. Spinal Nerve Ligation PIK-293 (SNL) (41) Briefly, the left L4 spinal nerve of mice, which is equivalent to L5 spinal nerve in PIK-293 rats (42), was exposed in isoflurane-anesthetized animals and tightly ligated between the DRG and their conjunction to form the sciatic nerve with a silk suture. Sham procedures were done in the same way without spinal nerve ligation. Behavioral tests were performed at designated times before collection of tissue samples, which were either processed immediately for biochemical studies or kept at ?80 C until use. Cav21 shRNA design and delivery via an adeno-associated viral vector (scAAV). A cDNA encoding the complete coding sequence of the mouse Cav21 subunit was obtained from Open Biosystems (IMAGE: 40061614), then cloned into a mammalian expression vector (pYFP-C1, Clontech). Candidate shRNAs were designed using publicly available web tools (Invitrogen Block-it and Genscript). These shRNAs were imbedded in a mir30 backbone using opposing BsmBI sites to insert complementary oligonucleotides encoding the shRNAs without altering the miR sequence (43, 44). The shRNAs were cloned into.