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Figure 2. Effects of HIF-1 activation on expression of 
-secretase components and proteolytic cleavage of APP and Notch. HeLa cells stably expressing human APP were treated with or without 1 mM NiCl2 for 2 or 4 h. A) Equal amounts of cell lysates were analyzed and immunoblotted with antibodies against HIF-1
, APH-1A, PEN-2, PS1-NTF, nicastrin, APP/APP CTFs, and -tubulin, respectively. B) The conditioned media were assayed for sAPP by immunoblotting with antibody (Ab) 6E10 and for Aß by immunoprecipitation using Ab 4G8 followed by Western blot analysis using 6E10. For assaying Notch, cells were transiently transfected with Notch
E construct and cell lysates were immunoblotted with anti-myc Ab 9E10 to detect Notch and NICD. The protein levels were quantified and normalized to controls. Note: For (A), the control was samples treated for 0 h; for (B), the control was the untreated samples at each time point. The ratios of NICD over Notch E were calculated and then normalized to controls. Data represent means ± SE from three separate experiments.
