RAW 264.7 cells were treated with IFNβ (50 U/ml) for the indicated times (to induce expression of TRAFD1). 25 μg total cell lysate was loaded on a 10 % polyacrylamide gel and blotted overnight at 4 °C with anti-TRAFD1 in 5 % milk at the indicated concentrations. Secondary HRP conjugated anti-Rabbit antibody was used at 1:10,000 dilution for 1 hr at room temperature. TRAFD1 is presumed to be the band just below 75 kDa.

RAW264.7 cells were treated with IFNβ (50 U/ml) for the indicated times (to induce expression of TRAFD1). Panel A, 1mg of cell lysate was incubated with 5 ug anti- TRAFD1 coupled to Protein-G-Sepharose beads overnight. 25ug of protein form the total cell lysate (TCL) or supernatant following immunoprecipitation (SN) was run on a 10 % polyacrylamide gel and probed using 1 μg/ml anti-TRAFD1 antibody at 4°C overnight followed by incubation with anti-Rabbit-HRP. Panel B Immunoprecipitated TRAFD1 was run on a 10 % polyacrylamide gel and probed using 1 μg/ml anti-TRAFD1 at 4 °C overnight followed by incubation with anti-Rabbit-HRP.