Fig. 7. Quantitation of pQC in Ifn-PrP transgenic mice.
(A) Expression of Ifn-PrP in mixed cortical cell cultures prepared from newborn transgenic and non-transgenic mice after treatment with proteasome inhibitor (10 μM MG132) for the indicated times. For comparison, PrP expression in normal hamster brain is shown. Detection was with the 3F4 monoclonal antibody selective to hamster (and not mouse) PrP. Two exposures of the blot are shown to illustrate the very low level steady state expression of Ifn-PrP, and the selective increase in the unglycosylated cytosolic form of PrP upon proteasome inhibition.
(B) Cortical cultures as in panel A were pre-treated with MG132 as indicated, pulse-labeled for 1 h with 35S-methionine in the absence or presence of MG132, and immunoprecipitated with either 3F4 (to selectively recover the transgenically expressed Ifn-PrP) or a pan-PrP antibody to recover both endogenous and transgenic PrPs. The white arrow indicates the position of unglycosylated (and non-translocated) Ifn-PrP, seen selectively when the proteasome is inhibited. This is also seen in the total PrP immunoprecipitates, where it represents ∼10% of total PrP synthesized.