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. Author manuscript; available in PMC: 2014 Feb 5.
Published in final edited form as: J Immunol. 2011 Apr 6;186(10):5638–5647. doi: 10.4049/jimmunol.1003801

FIGURE 4.

FIGURE 4

Tyk2-mediated phosphorylation-dependent polyubiquitination of TIEG1 is K27 linked. A, 293T cells were transfected with the indicated combination of plasmids encoding Flag-Tyk2, His-TIEG1, Myc-Itch, and HA-Ub for 48 h. Ubiquitinated TIEG1 was purified by Ni-NTA precipitation and analyzed by immunoblotting with anti-HA Ab. The expression of Tyk2 and Itch from total-cell lysates is shown (bottom two panels). B, 293T cells were transfected with plasmids encoding His-TIEG1, Myc-Itch, HA-Ub, and increased amounts of Flag-Tyk2. The ubiquitinated TIEG1 was purified by Ni-NTA precipitation and analyzed by immunoblotting with anti-HA Ab. C, 293T cells were transfected with plasmids encoding His-TIEG1, Myc-Itch, Flag-Tyk2, and HA-Ub or Ub-K48 or Ub-K63 (Ub-K48, only lysine K48 present, all other lysine mutated into arginine; Ub-K63, only lysine K63 present, all others mutated into arginine] for 40 h. Ubiquitinated TIEG1 was precipitated by Ni-NTA beads and analyzed by Western blotting with anti-HA Ab. The same blot was stripped and reprobed with anti-TIEG1. D, 293T cells were transfected with plasmids encoding His-TIEG1, Myc-Itch, Flag-Tyk2, and various lysine mutants of HA-ubiquitin (K0, all lysines are mutated into arginine; K6, K11, K27, K29, K33, K48, and K63, a single lysine present, all other lysines mutated into arginine). Ubiquitinated TIEG1 was precipitated and blotted with anti-HA Ab. E, 293T cells were transfected with Flag-Tyk2, Myc-Itch, and His-TIEG1 and HA-Ub WT, K0, K27, or K27R (K27R, only lysine K27 mutated into arginine, all other lysines present). The polyubiquitination of TIEG1 was determined by immunoblotting with anti-HA. F, 293T cells were transfected with Flag-Tyk2, Myc-Itch, HA-UbK27, His-TIEG1 WT, or His-TIEG1/Y179F mutant in 293T cells. The polyubiquitination of TIEG1 was determined by immunoblotting with anti-HA. The results shown are from one representative experiment that was conducted three times.