The antibody preparation and specificity tests are described in the Supplemental Experimental Procedures. The preparation of vesicles from synaptosomes and immunoblotting are described in the Supplemental Experimental Procedures. Levels of secreted FSTL1, CGRP, tenascin-C, SP, and glutamate were measured with immunoblotting, immunoassay, radioimmunoassay, or HPLC methods, respectively (see Supplemental Experimental Procedures). Cryostat sections of lumbar (L) 4–5 DRGs and spinal segments were immunostained. Postembedding immunogold

labeling and pre-embedding immunoperoxidase staining were performed. To distinguish the immunoperoxidase-labeled vesicles from unlabeled ones, we did not counterstain ultrathin sections with uranyl acetate and lead citrate. For axon immunostaining, the axons from L4–5 dorsal roots were fixed in −20°C methanol and double immunostained. The procedures selleck are provided in the Supplemental Experimental Procedures. COS7 cells were Selleck Gemcitabine cultured in DMEM containing 10% FBS. The cells were transfected with 1 μg plasmid and 10 μl Lipofectin Reagent (GIBCO) (30 mm dish) in serum-free medium for 6 hr and then in medium containing serum for 2–3 days. Neurons cultured from 60 DRGs were preincubated with FSTL1 for 30 min, and then stabilized with membrane-impermeant BS3 (Pierce)

for 60 min (see Supplemental Experimental Procedures). The cells were processed for immunoblotting or MS analysis (see Supplemental Experimental Procedures). The DRGs or COS7 cells were lysed and processed for immunoprecipitation and immunoblotting (see Supplemental Experimental Procedures). COS7 cells expressing NKA subunits or mutants and cultured DRG neurons were treated with

FSTL1 for 30 min. The NKA activity, which is the difference between total ATPase activity and ouabain-insensitive Mg2+-ATPase activity (Esmann, 1988), was measured many in the cell lysate (see Supplemental Experimental Procedures). The effect of FSTL1 was also studied with the NKA purified from the rat spinal cord (see Supplemental Experimental Procedures). The binding of 125I-FSTL1 to the transfected COS7 cells was measured (see Supplemental Experimental Procedures). The binding assay was performed by injecting synthetic peptides corresponding to each EL of the α1 subunit with a series of concentrations over the recombinant FSTL1 immobilized onto the sensor chip (Pharmacia BIAcore) (see Supplemental Experimental Procedures). The procedure for generating the conditional Fstl1 knockout mice is given in the Supplemental Experimental Procedures. Transverse spinal cord slices with an attached dorsal root from adult rats were prepared for blind whole-cell recording (see Supplemental Experimental Procedures). Dorsal root stimulation, which recruits Aδ- and C-fibers, was delivered with a suction electrode. Monosynaptic and polysynaptic eEPSCs were studied in the presence of 20 μM bicuculline and 2 μM strychnine (Nakatsuka et al., 2000).