Figure 3.—

Effect of coffee extract treatment on Aβ transgene expression and Aβ accumulation. (A) Quantitative RT–PCR of measurement of Aβ mRNA levels 24 hr after induction of CL4176 transgenic worms propagated on control or 10% coffee extract plates, average of three biologically independent experiments. (B) Anti-Aβ immunohistochemistry of anterior region of CL4176 worms induced for 24 hr after propagation on control or 10% decaffeinated coffee plates. Aβ immunoreactivity, red; DAPI counterstain, blue. Bar, 10 μm. (C) Immunoblot assay of Aβ accumulation 24 hr after upshift of CL4176 grown on control or 10% coffee extract plates. Left, visualization of monomeric (4 kDa band, boxed) and multimeric (higher molecular weight species) Aβ recovered from CL4176 worms induced for 24 hr after propagation on control or 10% decaffeinated coffee plates. Shown are total protein preparations (20 μg/lane) from four biologically independent experiments fractionated on a 4–12% polyacrylamide SDS gel and probed with anti-Aβ monoclonal antibody 6E10. The gel blot was reprobed with antibody against CstF-64 (Evans et al. 2001) to demonstrate equal lane loading. Right shows quantitation of average Aβ levels in coffee extract-treated CL4176 worms relative to untreated worms, obtained from the four independent experiments shown on the left. Total Aβ immunoreactivity (monomeric and multimeric bands) in each lane was quantified. Error bars = SEM.