FIG. 8.

Determinants of the association of the ΔID with Bcl10, TRAF6, and TAK1. (A) HEK293T cells were transfected with expression vectors for the indicated CARD11 variants in the absence (−) or presence (+) of the expression vector for FLAG-tagged Bcl10. Anti-FLAG immunoprecipitations were performed as described in Materials and Methods. To achieve approximately equivalent expression of each CARD11 variant, the following amount of each expression vector was used (in ng): wild type (WT), 100; ΔID, 100; ΔIDΔCARD, 1,000; ΔIDΔL1, 100; ΔIDΔCC, 100; ΔIDΔPDZ, 150; ΔIDΔL3, 125; ΔIDΔSH3, 150; ΔIDΔL4, 50; ΔIDΔGUK, 50. To achieve approximately equivalent expression of Bcl10 in each sample, 100 ng of Bcl10 expression vector was used in each sample except that 500 ng was used in the ΔIDΔCARD sample and 300 ng was used in the ΔIDΔPDZ sample. Western blotting (WB) with the indicated antibodies (at right) was performed to develop the contents of the immunoprecipitate (top and bottom panels) and the lysate input (middle panel). (B) Anti-FLAG immunoprecipitations were performed as described in panel A except that 200 ng of the expression vector for FLAG-tagged TRAF6 was used in all indicated samples (+). The following amount (in ng) of expression vector for each CARD11 variant was used: WT, 200; ΔID, 200; ΔIDΔCARD, 2,000; ΔIDΔL1, 200; ΔIDΔCC, 90; ΔIDΔPDZ, 300; ΔIDΔL3, 250; ΔIDΔSH3, 300; ΔIDΔL4, 100; ΔIDΔGUK, 100. (C) Anti-FLAG immunoprecipitations were performed as described in panel A except that 200 ng of the expression vector for FLAG-tagged TAK1 was used in all indicated samples (+). The following amount (in ng) of expression vector for each CARD11 variant was used: WT, 100; ΔID, 100; ΔIDΔCARD, 1,000; ΔIDΔL1, 100; ΔIDΔCC, 100; ΔIDΔPDZ, 150; ΔIDΔL3, 175; ΔIDΔSH3, 150; ΔIDΔL4, 50; ΔIDΔGUK, 50. α, anti; IP, immunoprecipitation.