For classical immunoprecipitations, indicated cells were washed once in phosphate-buffered saline and lysed in IP buffer. Protein lysates were incubated overnight with 5 μL of Flag or V5 antibody-conjugated beads (Sigma-Aldrich) or with 1 μl of p50 antibody together with 20 μl of Protein A magnetic beads (Life Technologies). After 4 washes in IP buffer, the beads were resuspended in 2X Laemmli's buffer and subjected to immunoblotting analysis.
For immunoprecipitation of CD95 complexes, HEK/293T cells were transfected with Flag-KPC2 or Flag-p65. After 24 h, transfected cells were lysed in Hepes Buffer (Hepes 25 mM, NaCl 150 mM, NaF 2 mM, NaVO4 1 mM, EGTA 2 mM). Cell lysates were incubated for 2 h at 4°C with 100 ng of the different GST-V5-CD95 constructs and then overnight at 4°C with 10 μl of Anti-V5 Agarose Beads (Sigma-Aldrich). After extensive washing in Hepes buffer, the precipitated complex was resolved by SDS-PAGE and immunoblotting was performed with the indicated antibodies.
For immunoprecipitation of CD95 complexes, HEK/293T cells were transfected with Flag-KPC2 or Flag-p65. After 24 h, transfected cells were lysed in Hepes Buffer (Hepes 25 mM, NaCl 150 mM, NaF 2 mM, NaVO4 1 mM, EGTA 2 mM). Cell lysates were incubated for 2 h at 4°C with 100 ng of the different GST-V5-CD95 constructs and then overnight at 4°C with 10 μl of Anti-V5 Agarose Beads (Sigma-Aldrich). After extensive washing in Hepes buffer, the precipitated complex was resolved by SDS-PAGE and immunoblotting was performed with the indicated antibodies.