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. 2004 Jan 8;23(2):322–332. doi: 10.1038/sj.emboj.7600044

Figure 1.

Figure 1

Intact RING and zinc finger domains of TRAF2 are required for its ubiquitination in vivo. (A) In vivo ubiquitination of endogenous TRAF2. HeLa cells were treated with hTNFα (40 ng/ml) at indicated time points, and proteins were extracted with RIPA buffer (supplemented with 20 mM NEM and 5 mM ubiquitin aldehyde). Protein extracts were then subjected to immunoprecipitation with anti-TRAF2 Ab or control rabbit IgG, followed by immunoblot analysis with the aid of anti-ubiquitin Ab (upper panel; ns: nonspecific bands). The same membrane was stripped and reprobed with anti-TRAF2 Ab (lower panel). (B) Intact RING and zinc finger domains of TRAF2 are required for its ubiquitination in vivo. Flag-TRAF2 (1 μg), Flag-TRAF2-Rm (1 μg, C49A, H51A, C54A and C57A), Flag-TRAF2-RmZm (1 μg, C49A, H51A, C209A and C212A) and Flag-ΔN-TRAF2 (1 μg, Δ1–87 amino acids from N-terminal) were co-transfected with HA-Ub (2 μg) to HeLa cells. At 36 h after transfection, proteins were extracted with 2% SDS/TBS, diluted with 9 vol 1% Triton X-100/TBS and subjected to immunoprecipitation (IP) using anti-Flag Ab and Western blot analysis using anti-HA Ab. The same membrane was stripped and reprobed with anti-TRAF2 polyclonal Ab (lower panel).