Ubiquilin 1 knock-down regulates APP-Gal4 metabolism in SY5Y-APP-Gal4 cells. (A) Ubiquilin 1 knock-down decreases AICD-Gal4-mediated luciferase activity. SY5Y-APP-Gal4 cells stably expressing the control shRNA, an APP specific shRNA, or five different shRNA targeting Ubiquilin 1 were generated. Cell lysates were utilized to measure AICD-Gal4 mediated luciferase activity. Bars represent the mean normalized luciferase activity (+/- standard error) of six independent trials. (B) SY5Y-APP-Gal4 cells stably expressing Ubiquilin 1 specific shRNA (#2) have decreased Ubiquilin 1, mature and immature APP-Gal4, C83-Gal4, AICD-Gal4, and sAPPα levels compared to cells expressing control shRNA. (C) Quantification of Western blot results. Bars represent mean densitometry (+/- standard error) of three independent trials. Black bars represent the densitometry from Ubiquilin 1 knock-down cells; white bars represent the densitometry from cells expressing control shRNA. Abbreviations: Ma APP denotes mature APP, Im APP denotes immature APP; Ma/Im APP denotes the mature APP/immature APP ratio. Statistical significance between mock and over-expression for each measure was determined using a two-sample, one tailed t-test and sequential Bonferroni procedure to adjust for multiple comparisons. * indicates p < 0.05; ** indicates p < 0.01. (D) Ubiquilin 1 knock-down does not alter APP mRNA levels compared to control shRNA as measured by quantitative PCR.