Figure 1. Mature forms of APP and BACE1... | Rockefeller University Press

Figure 1.

$Figure 1. Mature forms of APP and BACE1 show association with Lubrol-resistant membranes. (a) Mature forms of APP were efficiently recovered in DRM fractions derived from mouse brains solubilized with Lubrol WX but poorly with Triton X-100 or Brij 97. All three detergents showed recovery of PrP (a DRM marker) in DRM fractions well separated from calnexin (a detergent-soluble membrane marker). Arrows, different forms of FL-APP; m, mature forms; im, immature form of APP. (b) Quantitative comparison of distributions of mature and immature forms of APP across the gradients after Lubrol WX or Brij 97 solubilization. Each point represents a percentage of the total. Results are mean ± SD based on three to four independent experiments. (c) Mature forms and CTFs of APP and BACE1 associate with DRM fractions derived from primary cultured cortical neurons solubilized with Lubrol WX, whereas ADAM17 mainly resides in Lubrol-soluble membranes. GPI-anchored proteins Thy-1 and PrP were used as DRM markers, and calnexin and GM130 were used as detergent-soluble membrane markers. We used longer exposure to detect APP-CTFs compared with FL-APP. (d) Lubrol WX causes only negligible levels of artifactual intermingling of Thy-1. Rat and mouse brain tissues were cohomogenized in 1% Lubrol WX or 1% Brij 97, and DRMs were prepared. Thy-1 on DRMs was immunoaffinity purified and probed with species-specific antibodies. Lubrol caused only negligible levels of mixing of Thy-1 on DRMs during solubilization, centrifugation, and immunoisolation.$

Mature forms of APP and BACE1 show association with Lubrol-resistant membranes. (a) Mature forms of APP were efficiently recovered in DRM fractions derived from mouse brains solubilized with Lubrol WX but poorly with Triton X-100 or Brij 97. All three detergents showed recovery of PrP (a DRM marker) in DRM fractions well separated from calnexin (a detergent-soluble membrane marker). Arrows, different forms of FL-APP; m, mature forms; im, immature form of APP. (b) Quantitative comparison of distributions of mature and immature forms of APP across the gradients after Lubrol WX or Brij 97 solubilization. Each point represents a percentage of the total. Results are mean ± SD based on three to four independent experiments. (c) Mature forms and CTFs of APP and BACE1 associate with DRM fractions derived from primary cultured cortical neurons solubilized with Lubrol WX, whereas ADAM17 mainly resides in Lubrol-soluble membranes. GPI-anchored proteins Thy-1 and PrP were used as DRM markers, and calnexin and GM130 were used as detergent-soluble membrane markers. We used longer exposure to detect APP-CTFs compared with FL-APP. (d) Lubrol WX causes only negligible levels of artifactual intermingling of Thy-1. Rat and mouse brain tissues were cohomogenized in 1% Lubrol WX or 1% Brij 97, and DRMs were prepared. Thy-1 on DRMs was immunoaffinity purified and probed with species-specific antibodies. Lubrol caused only negligible levels of mixing of Thy-1 on DRMs during solubilization, centrifugation, and immunoisolation.

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