Figure 2.

Increased ubiquitin precedes activation of ER stress. A, Representative Western blot of pPERK, BiP, ubiquitin, tau, and actin from iHEK-Tau cells induced to express tau by adding Tet to the media for 4 d. Samples were collected at the indicated times. B, Linear regression analysis representing the relative quantitation of pPERK, BiP, and ubiquitin levels in A over time; values are relative and normalized to GAPDH and tau. Significance was derived from four experiments; *p < 0.05, **p < 0.01, ***p < 0.001 for C, E, G. Representative immunofluorescent images of Tg hippocampi of the indicated ages and stained for ubiquitin (C; green), pPERK (E; red), and tau (G; red). Scale bar, 100 μm. D, F, H, Quantitative analysis of C, E, and G. Immunofluorescence in 3-month-old (3MO) sections was virtually undetectable above background control slide, which consisted of immunofluorescent staining without primary antibody incubation. Compared with 6MO mice, ubiquitin, pPERK, and tau levels increased by 150% (9MO; †p < 0.0001) and 186% (12MO; †p < 0.0001), 266% and 243% (9MO; †p < 0.0001), and 194% (9MO; ***p < 0.001) and 215% (12MO; ***p < 0.001), respectively. Comparing 9MO with 12MO mice, ubiquitin and tau levels were significantly different (†p < 0.0001 and ***p < 0.001, respectively), but pPERK levels were not (p > 0.05) (n = 3 mice for each condition). Statistical significance was determined using Student's t test.