RAW264.7 cells were treated with IFNβ for the indicated times (to induce expression of TRAFD1) and with the indicated concentrations. 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-Sheep antibody was used at 1:10,000 dilution for 1 h at room temperature. Bleeds 1, 2 and 3 performed similarly. The above image is from bleed 1.
RAW264.7 cells were stimulated with IFNβ for 8 h or left unstimulated as indicated. Cells were lysed and total cell lysate (lanes 1 and 2) was run alongside supernatants after immunoprecipitation of 1 mg cell lysate with 0.5, 1, or 2 μg anti-TRAFD1 (lanes 3-8) or 2 μg of sheep pre-immune IgG (lanes 9 and 10) coupled to ProteinG-Sepharose beads. 25 μg of lysate was run in each case. The above image is from bleed 1.