Fig. 2

ERp57 overexpression reduces mutant SOD1 aggregation at the disease end-stage. a NSC-34 cells were co-transfected with constructs to express SOD1^G93A-EGFP or SOD1^G93A-EGFP tagged to the ER (ER-SOD1^G93A) and ERp57-V5 tag or empty vector (mock). Filter-trap analysis under non-reducing (−DTT) and reducing (+DTT) conditions was performed 48 h after transfection for detection of SOD1 aggregates. Total SOD1 levels were assessed using western blot analysis under reducing (+DTT) conditions. Control: cells transfected with empty vector for SOD1 constructs. b Quantification of a. Results are plotted as fold change relative to mock transfected cells (dotted line). β-actin was employed as loading control. Statistical analysis was performed using Student’s t-test to compare against mock transfected cells. Mean ± S.E. is shown; p values: n.s., p > 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001 (n = 6 independent experiments). c Filter-trap analysis of end-stage lumbar spinal cord extracts under non-reducing (−DTT) and reducing (+DTT) conditions for detection of SOD1 aggregates. β-actin and SOD1 western blot were employed as loading controls. d Quantification of c. DTT resistant aggregates were calculated directly from +DTT signal. DTT-sensitive aggregates were calculated as –DTT minus +DTT signal. Statistical analysis was performed using Student’s t-test. Mean ± S.E. is shown; p values: n.s., p > 0.05 and *, p ≤ 0.05 (n = 8–11 animals per genotype). e Filter-trap analysis of post-natal day 90 lumbar spinal cord extracts under non-reducing (−DTT) and reducing (+DTT) conditions to detect SOD1 aggregates at early-symptomatic stage. β-actin and SOD1 western blots were employed as loading controls. f Quantification of e. DTT resistant aggregates were calculated directly from +DTT signal. DTT-sensitive aggregates were calculated as –DTT minus +DTT signal. Statistical analysis was performed using Student’s t-test. Mean ± S.E. is shown (n = 4–10 animals per genotype). For c and e, each lane and dot corresponds to one animal