Fig. 1. (A) Siah2 targets the ubiquitylation of TRAF2 in vitro. Bacterially expressed and purified GST–Siah2 was subjected to an in vitro ubiquityl ation reaction in the presence of E1 and/or E2. The lower panel depicts Ponceau S staining of the blot. (B) Bacterially expressed and purified GST–TRAF2 in its wild-type, RING mutant (G-Rm-TRAF2; C74A, H76A) or RING-deleted (G-ΔN-TRAF2; Δ1–87aa) forms were subjected to an in vitro ubiquitylation reaction by a soluble form of bacterially expressed and purified Siah2. Following the reaction, GST–TRAF2 was subjected to extensive washing with PBS buffer containing empigen BB (1%) NP-40 (0.5%), LiCl (0.5 M) β-ME (0.1%) and EDTA (2 mM). Ubiquitylation of TRAF2 was detected using anti-HA antibody. The lower panel depicts Ponceau S staining of GST–TRAF2 used for the reaction. (C) Bacterially expressed and purified His-TRAF2 and Siah2 in solution were subjected to an in vitro ubiquitylation reaction followed by immunoprecipitation of TRAF2 using anti-TRAF2 antibody. After extensive washing, ubiquitylation of TRAF2 was detected using anti-HA antibody. The middle panel depicts the amount of TRAF2 immunoprecipitated in these reactions. The lower panel depicts immunoblot analysis of Siah2, which was performed on the supernatant (material that was not immunoprecipitated by TRAF2 antibodies). (D) In vitro translated and ³⁵S-labeled TRAF2 (in reticulocyte lysates that were immunodepleted of Siah2) was subjected to an in vitro ubiquitylation/degradation reaction, which was carried out in the presence of E1, E2 and GST–Siah2 for the indicated times. The degree of degradation was assessed by direct assessment of the radioactive signal from each lane. The degree of ubiquitylation is shown as marked in the right panel. (E) In vitro translated and ³⁵S-labeled TRAF2 was subjected to an in vitro ubiquitylation/degradation reaction as indicated in (D), except that RING mutant Siah2 was also added to the reaction as indicated. (F) In vitro translated and ³⁵S- labeled TRAF2 or Siah2 were incubated with GST, GST–Siah2 or GST–TRAF2 on beads for 1 h at 4°C followed by four washes with buffer A or B (see Materials and methods for details), subsequent separation by SDS–PAGE and detection of bound [³⁵S]TRAF2 or [³⁵S]Siah2.