Poly(I:C) promotes TNFa/TNFR1-dependent oligodendrocyte death in mixed glial cultures

Steelman, Andrew J; Li, Jianrong

Cell lysates from microglia, astrocyte and oligodendrocyte monocultures as well as splenocytes were used to determine the presence of TLR3 protein. Briefly, cells were lysed on ice in standard lysis buffer (10 mM Na 2 P 2 O 7 , 10 mM NaF, 100 mM Na 3 VO 4 100 mM PMSF proteinase inhibitor cocktail; Roche) and then sonicated. Cell debris was pelleted by centrifugation at 16000 × g for 15 min at 4°C. Total proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis followed by transferring to PVDF membranes. After blocking with TBS-T (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat milk for 1 h at RT the membranes were incubated with anti-TLR3 (1: 200, Rabbit IgG, Santa Cruz Biotech, Santa Cruz, CA.) overnight at 4°C or β-actin (1:10,000, Mouse IgG, Sigma, St. Louis, MO) diluted in TBS-T containing 5% non-fat milk. The membranes were washed 3-5 times with TBS-T, and then incubated with horseradish peroxidase conjugated secondary antibody for 1 h. Following additional washes bands were detected by chemiluminescence using the SuperSignal detection kit (Thermo Scientific, Rockford, IL).