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. 2014 Oct 31;34(1):4–19. doi: 10.15252/embj.201488896

Figure 3. Secreted ERdj3 inhibits extracellular protein aggregation.

Figure 3

  1. A Immunoblot of conditioned media collected from HEK293T cells transiently transfected with ERdj3WT or ERdj3H53Q and incubated with an affinity resin consisting of native (N) or guanidinium hydrochloride (GdnHCl)-denatured (D) RNase A covalently coupled to sepharose resin. Resin was incubated with media for 2 h at ambient temperature, followed by washing and then elution in Laemmli reducing buffer.
  2. B Plot showing the time-dependent increase in ThioT fluorescence (ex: 440 nm, em: 485 nm) of Aβ1–40 incubated (10 μM, 37°C, pH 7.2) with regular agitation in the presence or the absence (black) of recombinant ERdj3 (RERdj3). The RERdj3 concentrations and molar ratios of RERdj3 to Aβ1–40 are as follows: violet: 1.5 nM, 1:6,561; light blue: 4.6 nM, 1:2,187; dark blue: 14 nM, 1:729; dark green: 41 nM, 1:243; light green: 120 nM, 1:81; yellow: 370 nM, 1:27; orange: 1.1 μM, 1:9; red: 3.3 μM, 1:3, or maroon: 10 μM, 1:1. All traces represent the average of three replicates.
  3. C Plot showing the time-dependent increase in ThioT fluorescence of Aβ1–40 incubated (10 μM, 37°C, pH 7.2) with regular agitation in the presence or the absence of BSA (14 μg/ml) or RERdj3 (14 μg/ml; 370 nM, RERdj3: Aβ = 1:27). All traces represent the average of three replicates.
  4. D Immunoblot of Aβ1–40 incubated (10 μM, 37°C, pH 7.2) with regular agitation for 90 h in the presence or the absence of RERdj3 (14 μg/ml; 370 nM, RERdj3: Aβ1–40 = 1:27) or BSA (14 μg/ml). The reaction solution in the well was collected (Nonadhered) and separated into soluble and insoluble fractions by centrifugation. These samples, as well as the washed wells of the plate (Adhered), were denatured in 8 M GdnHCl with sonication and analyzed by SDS-PAGE/immunoblotting.

Source data are available online for this figure.