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. 2001 Oct 16;98(22):12654–12658. doi: 10.1073/pnas.231471798

Figure 4.

Figure 4

dMyD88 associates with Toll and Pelle. (A) S2 cells were transiently transfected with dMyD88 (176) tagged with a V5 epitope and Toll 10b. Cell lysates were incubated overnight with Protein G Sepharose and either Toll-specific rabbit immune serum or rabbit serum of irrelevant specificity. Immunoprecipitates were analyzed by SDS/PAGE immunoblot with a V5-specific antibody. dMyD88 (176) can be detected in anti-Toll immunoprecipitates (Top, lane 2) but not in control immunoprecipitates from cells transfected with dMyD88 alone (Top, lane 1) or in controls immunoprecipitated with the irrelevant antiserum (Top, lanes 3 and 4). Whole cell lysates (50 μg/lane) immunoblotted with V5-specific antibody (Bottom) and Toll-specific antiserum (Bottom) confirm expression of the indicated proteins. dMyD88 therefore can be detected in active Toll receptor complexes and the middle, TIR, and C-terminal domains are sufficient for association of dMyD88 with Toll. (B) S2 cells were transfected with dMyD88 (1) tagged with a V5 epitope and Flag-tagged Pelle. Lysates were incubated overnight with anti-Flag M2 agarose beads, and immunoprecipitates were analyzed by SDS/PAGE followed by immunoblotting with V5 antibody and Flag antibody. dMyD88 (1) coimmunoprecipitated with Flag Pelle (Top, lane 3). No dMyD88 (1) could be detected in immunoprecipitated lysates of untransfected cells (Top, lane 1) or cells transfected with dMyD88 (1) alone (Top, lane 2). Immunoblotting of whole cell lysates show appropriate expression of proteins (bottom two sections). The death domain and middle domain of dMyD88 are therefore sufficient for interaction with Pelle.