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. 2007 Oct 10;2(10):e1030. doi: 10.1371/journal.pone.0001030

Figure 2. Ubiquitination of mutant SOD1 aggregates.

Figure 2

(A–R) Colocalization assay with SOD1 and ubiquitin. SK-N-SH cells expressing wild-type SOD1 (A–I) or L84V SOD1 (J–R) were incubated with 1 µg/ml of tunicamycin (D–F, M–O), 4 µg/ml of ALLN (G–I, P–R), or no agents (A–C, J–L) for 24 h. Then the cells were fixed and stained with anti-SOD1 antibody (green; A, D, G, J, M, P) or anti-ubiquitin antibody (red; B, E, H, K, N, Q). Arrows indicate colocalization of SOD1 aggregates and ubiquitin. Scale bar = 20 µm. (S) Co-immunoprecipitation assay utilizing ubiquitin. SK-N-SH cells stably expressing wild-type and L84V SOD1 were transfected with a myc-tagged ubiquitin expression vector. After incubation with or without ALLN, cell lysates were prepared and assayed with anti-myc antibody of the immunoprecipitant with anti-FLAG antibody. Asterisk shows an ubiquitinated ladder that appeared after ALLN treatment of L84V SOD1-expressing cells. IgG bands are shown as loading controls.