Figure 2
Disulfiram increases ADAM10 transcription dose-dependently and leads to enhanced ADAM10 protein amount and activity. SH-SY5Y cells were transiently transfected with a reporter vector for human ADAM10 (hADAM10) (a) murine ADAM10 (mADAM10) (b) or the empty vector as a negative control. Subsequently, cells were incubated with disulfiram in the indicated concentration or DMSO as a solvent-control (values set to 100%). The experiment was conducted three-times independently in duplicates, values were normalized to protein content of the cell lysate and are represented as mean + SD. Statistical analysis: One Way ANOVA with Tukey’s multiple comparisons test; ***p < 0.001; **p < 0.01; *p < 0.05; ns, p > 0.05). (c) For protein analysis, SH-SY5Y cells were incubated for 48 h with disulfiram as indicated or DMSO as solvent-control. Cell lysate adjusted for protein content was used for quantitation of ADAM10, full-length APP (antibody against APP C-terminus) and GAPDH; culture supernatants were analyzed in regard to sAPP-alpha (antibody 6E10, directed against the N-terminus). Representative blots are shown. Blots were cropped for combining the figure, single blot parts are divided by a white space. For full-length blot pictures please see Suppl. Figure 1. (d) Values obtained by densitometrical analysis for the specific membrane-tethered proteins were normalized to values obtained for GAPDH for quantitative analyses. Experiments were performed at least three-times independently in duplicate (n ≥ 6). Statistical analysis: One Way ANOVA *p < 0.05).
