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. 2016 Sep 6;291(43):22650–22660. doi: 10.1074/jbc.M116.733451

FIGURE 2.

FIGURE 2.

TIPE2 interferes with the binding of TAB1 and TAB2 with TAK1, which inhibits phosphorylation and ubiquitination of TAK1 and its activity. A and B, HEK293T cells were transfected with 6× Myc-TIPE2 alone or the indicated combination of 6× Myc-TIPE2, FLAG-TAK1, and HA-TAB1 (A) or HA-TAB2 (B). After 24 h, the cells were extracted and immunoprecipitated using an anti-FLAG antibody. Binding of TAB1 or TAB2 with TAK1 was detected by Western blotting assay using an anti-HA antibody. Expression of FLAG-TAK1, 6× Myc-TIPE2, HA-TAB1, and HA-TAB2 in the immunoprecipitates was detected by Western blotting assay using the appropriate anti-tag antibody. C and D, HEK293T cells were transfected with HA-TIPE2 alone or the indicated combination of HA-TIPE2, FLAGTAK1, and 6× Myc-TAB1 (C) or 6× Myc-TAB2 (D). After 24 h, the cells were extracted and immunoprecipitated using an anti-FLAG antibody. The phosphorylation of TAK1 was detected by Western blotting assay using an anti-pTAK1 antibody. The expression of FLAG-TAK1, HA-TIPE2, 6× Myc-TAB1, and 6× Myc-TAB2 in the immunoprecipitates was detected by Western blotting assay. E, HEK293T cells were transfected with the indicated combination of FLAG-TAK1, Halo-TIPE2, Halo-vector, HA-K63-Ub, and 6× Myc-TAB1 vectors. After 24 h, whole cell lysates were extracted and immunoprecipitated using an anti-FLAG antibody. The ubiquitination of TAK1 was detected by Western blotting assay using an anti-HA antibody. The expression of FLAG-TAK1, Halo-TIPE2, and 6× Myc-TAB1 in whole cell lysates was detected by Western blotting assay. F, HEK293T cells were transfected with the indicated combination of HA-TIPE2, FLAG-TAK1, and 6× Myc-TAB1 vectors. After 24 h, the cells were extracted, and then TAK1 activity in the whole lysates was monitored by detecting phosphorylation of MKK3/6, a substrate of TAK1, by Western blotting assay using an anti-pMKK3/6 antibody. The expression of FLAG-TAK1, HA-TIPE2, 6× Myc-TAB1, MKK6, and phosphorylation of MKK3/6 in whole lysates was detected by Western blotting assay using the appropriate anti-tag antibody, anti-MKK6, and anti-pMKK3/6 antibody. IB, immunoblot; IP, immunoprecipitation.