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. 2005 Oct 27;24(22):3859–3868. doi: 10.1038/sj.emboj.7600845

Figure 5.

Figure 5

NRIF is K63-polyubiquitinated. (A) HEK cells were transfected with Flag-NRIF and either with HA-tagged WT ubiquitin, K29R, K48R or K63R point mutants of ubiquitin. The cells were then lysed in SDS lysis buffer, equal amounts of lysate were immunoprecipitated with NRIF antibody and immunoblotted with anti-Flag to detect NRIF (upper panel) or anti-ubiquitin (middle panel). The lysates were Western blotted with anti-HA to examine the expression of HA-tagged ubiquitin mutants (lower panel). (B) PC12 cells were transfected with HA-tagged WT, K29R, K48R or K63R ubiquitin mutants. The cells were treated with Δ9/13 NGF mutant for 5 min and lysed with SDS lysis buffer. NRIF was immunoprecipitated and Western blotted with anti-HA to examine the ubiquitination of NRIF (upper panel) or with anti-NRIF (middle panel) and the lysates were blotted with anti-HA (lower panel). (C) HEK 293 cells were transfected with Flag-NRIF, the cells lysed, then NRIF immunoprecipitated and the in vitro ubiquitination assay was carried out in the presence or absence of WT ubiquitin, a mutant with only K63 available for forming the ubiquitin chain (K63 Ub), or the K63R mutant ubiquitin along with E1, UbcH7 (E2) and TRAF6 (E3). This was followed by addition of 5 μg of GST-UBA in binding buffer to the above complex; the NRIF beads were then washed and subjected to Western blot analysis for ubiquitin, NRIF or GST (as indicated). Note that the GST-UBA only precipitated with the ubiquitinated form of NRIF. (D) HEK 293 cells were transfected with Flag-NRIF, myc-tagged WT p62, a p62 construct lacking the N-terminal 229 residues (p62-ΔN-term), or a p62 mutant lacking the UBA domain (p62-ΔUBA) along with Flag-TRAF6 or HA-Ub. Cells were then lysed in Triton lysis buffer, NRIF immunoprecipitated and the precipitates were immunoblotted with antibody to NRIF, TRAF6 or the myc-tag as indicated (upper panels). The lysates (50 μg) were subjected to Western blot analysis with anti-NRIF, anti-TRAF6 or anti-myc to verify the expression levels of NRIF, TRAF6 and p62 (lower panels).