Figure 3.
SOD1mut attenuates the retro-translocation of ERAD substrate on the components of ERAD. (A) Lysates from HEK293 cells, transfected with Derlin-1-HA, Myc-VIMP, and Flag-SOD1 at the indicated combinations, were analyzed by IP-IB. The presence of Derlin-1-HA and Flag-SOD1 in the same lysates is shown. (B) Lysates from HEK293 cells, transfected at the indicated combinations, were analyzed by IP-IB. The presence of p97 and Myc-SOD1 in the same lysates is shown. (C) Lysates from HEK293 cells, transfected at the indicated combinations, were analyzed by IP-IB. The presence of Derlin-1-HA and Myc-SOD1 in the same lysates is shown. (D) Lysates from HEK293 cells, transfected with NHK, Derlin-1-HA, Myc-VIMP, and Flag-SOD1 at the indicated combinations, were analyzed by IP-IB. The presence of Derlin-1-HA, Myc-VIMP, and Flag-SOD1 in the same lysates is shown. (E) HEK293 cells were transfected with NHK-Flag, Derlin-1-HA, Myc-SOD1, and HA-Ub at the indicated combinations and incubated with 0.25 μM MG132 for 18 h. NHK was immunoprecipitated with antibody to Flag. After incubation with the denaturing buffer containing 1% SDS, NHK was reimmunoprecipitated with antibody to Flag. Samples were immunoblotted with antibodies to HA and Flag. The presence of Derlin-1-HA and Myc-SOD1 in the same lysates is shown. Asterisks denote nonspecific bands and IgG.