Figure 6: GRP78 reduces the amount of protease-resistant PrPSc in vitro.
From: The Endoplasmic Reticulum Chaperone GRP78/BiP Modulates Prion Propagation in vitro and in vivo
(A) RML infected brain homogenates were incubated with different concentrations of purified recombinant GRP78 for 20 h (1200 minutes), and the amount of PrPSc remaining resistant to PK digestion was assessed by Western blotting (top panel). Numbers at the top represent GRP78 concentrations, expressed as μM. (B) Highly purified PrPSc preparations were incubated with different concentrations of GRP78 for 20 h, and reactions were analyzed by Western blotting. As a control, BSA (bovine serum albumin) was used at the same concentrations. Numbers at top of each gel represent μM protein concentration supplemented in each case. Graphs below each blot represent the densitometric analysis of 3 replicates for each respective experiment. Values correspond to the average ± standard error and differences were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. (C) Purified PrPSc preparations were incubated with 8 μM of GRP78 for different time points ranging from 30 to 1200 minutes. “Control” represents a purified PrPSc aliquot without any treatment. The graph below represents the densitometric analysis of 3 replicates showed as the average ± standard error. Differences were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. All samples used to evaluate PrPSc signal were first treated with PK. Immunoblot was used to assess remaining protease-resistant PrPSc in each case. *P < 0.05; ***P < 0.001.
