Cntfr

Frozen brain sections or neurons (4mm) cultured on glass coverslips were xed with 4% paraformaldehyde for 30min and then treated with 0.1% Triton X-100 for 10 min at room temperature. Subsequently, the sections were blocked with 5% normal goat serum in phosphate-buffered saline (PBS) for 1 h at room temperature. Then, the sections were incubated overnight at 4°C with the following primary antibodies: TSP1, TNFR1, FZD2, CNTFR, spinophilin, and MAP2 (Abcam) that were detected by incubation for 30 min with uoresceine isothiocyanate (FITC) (green)/Alexa Fluor 594 (red)-conjugated secondary antibody. The images were captured using a Leica TCS SP2 (Leica Microsystems, Heerbrugg, Switzerland) confocal laser scanning microscope. The imaging data were analyzed and quanti ed using Image Pro Plus software.

The total amount of protein in the lysates was determined by the BCA protein assay (Amresco,USA). An equivalent of 50 µg protein was resolved by 10% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and electroblotted to Polyvinylidene Fluoride (PVDF) membrane that was blocked with 5% non-fat dry milk in Tris buffer saline and Tween 20 (TBST) (150 mM NaCl, 50 mM Tris, 0.05% Tween 20). Subsequently, the membranes were probed with primary antibodies, TSP1, p38MAPK, TNFα, TNFR1, Wnt7a, frizzled2 (FZD2), CNTF, CNTFR, spinophilin, and β-actin (Abcam Cambridge, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody(Abcam, Cambridge, UK). After extensive washing, the immunoreactive bands were visualized by ECL reagent (Thermo USA) after exposure on Kodak BioMax lm (Kodak). The band intensities were quanti ed using QuantityOne software. The intensities were expressed as fold-change relative to the GAPDH levels.
For coimmunoprecipitation, the lysates of tissues were incubated with antibodies overnight (4°C) and subsequently with protein G-agarose beads (Millipore, USA) for 5 h (4°C). Followed by washes with lysis buffer, the eluent was separated by SDS-PAGE and electroblotted to PVDF membrane using primary and secondary antibodies.