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

Figure 7.

Figure 7

K63 polyubiquitinated NRIF is transported to the nucleus. (A) HEK 293 cells were transfected with Flag-NRIF, myc-tagged p75 along with HA-K48R or K63R ubiquitin mutants. The cells were then treated with 50 ng/ml NGF for 5 min, the cytosolic and nuclear fractions were immunoprecipitated with NRIF antibody and Western blotted using an NRIF antibody. The cytosolic and nuclear fractions were also immunoblotted for CRM1 and actin to validate the purity of the fractions (left panels). The total lysates were Western blotted using anti-NRIF, p75 or HA to confirm the expression of NRIF, p75 and ubiquitin constructs (right panels). (B) PC12 cells were transfected with the HA-tagged K48R or K63R ubiquitin mutant, then stimulated with Δ9/13 NGF (50 ng/ml) for 5 min, and cytosolic and nuclear fractions were isolated. NRIF was immunoprecipitated from these fractions and immunoblotted with anti-NRIF. The cytosolic and nuclear fractions were also immunoblotted for CRM1 and actin to validate the purity of the fractions (left panels). The expression of the NRIF and HA-tagged ubiquitin mutants was verified by Western blot with anti-NRIF and anti-HA (right panels).