Figure 1.

SOD1^mut triggers ER stress and inhibition of ERAD. (A) NSC34 cells were lysed after infection with Ad-SOD1^wt, Ad-SOD1^A4V, Ad-SOD1^G85R, or Ad-SOD1^G93A for 48 h or treatment with 2.5 μg/mL tunicamycin (a potent inducer of ER stress) for 2 h and analyzed by immunoprecipitation-immunoblotting (IP-IB) with antibodies to IRE1α or PERK. (P-IRE1) Activated IRE1; (P-PERK) activated PERK. The presence of SOD1 in the same lysates is shown. (B) NSC34 cells were transfected with SOD1^wt or SOD1^mut for 48 h. Expression of BiP and CHOP was examined by RT–PCR. (C) NSC34 cells were transfected with NHK and SOD1^wt or SOD1^mut. Cells were pulse-labeled with [³⁵S] methionine and cysteine and chased for the indicated time periods. Cell lysates were immunoprecipitated with antibody to α1AT. (NHK + CHO) Glycosylated NHK; (NHK − CHO) deglycosylated NHK. The relative radioactivities in NHK at different times of chase were calculated and are shown as fold decreases relative to the intensity observed at 0 h chase. (D) NSC34 cells were transfected with Venus^U and SOD1^wt or SOD1^mut. Cells were pulse-labeled with [³⁵S] methionine and cysteine and chased for the indicated time periods. Cell lysates were immunoprecipitated with antibody to GFP. The relative radioactivities in Venus^U at different times of chase were calculated and are shown as fold decreases relative to the intensity observed at 0 h chase.