Figure 2. The α-secretases A10 and TACE interact with γ-secretase at overexpressed levels and at the plasma membrane. (A) CHAPSO-solubilized lysates of γ-30 CHO cells were immunoprecipitated for Aph-1 or PAA as a control in the absence or presence of the metalloprotease inhibitor 1,10-phenanthroline. Immunoprecipitates were blotted to probe for coIP of A10 or TACE, APP, and for the γ components NCT and Pen-2. (B) γ-30 lysates were immunoprecipitated for either A10 or NPR A as a control. Resulting immunoprecipitates were probed for the coIP of PS1 CTF. (C) Lysates from cells that express A10 only, human γ-secretase only, neither, or both (lanes 1, 2, 3, and 4, respectively) were specifically pooled to form mix 1 (A10 only + γ-secretase only) or mix 2 (neither + both) and then immunoprecipitated for γ-secretase with an M2 resin targeting the Flag tag on Pen-2. Immunoprecipitates were probed for the coIP of A10 and the γ components PS1 NTF and Pen-2. (D) γ-30 cells were treated with a nonpermeable biotinylation reagent at 4°C to label cell surface proteins. Lysates were immunoprecipitated for Aph-1, and the resultant immunoprecipitates were eluted and pulled down with streptavidin to enrich for cell surface interactors of γ-secretase, followed by blotting for α- and γ-secretases as in A. im, immature; m, mature.
Figure 2.

The α-secretases A10 and TACE interact with γ-secretase at overexpressed levels and at the plasma membrane. (A) CHAPSO-solubilized lysates of γ-30 CHO cells were immunoprecipitated for Aph-1 or PAA as a control in the absence or presence of the metalloprotease inhibitor 1,10-phenanthroline. Immunoprecipitates were blotted to probe for coIP of A10 or TACE, APP, and for the γ components NCT and Pen-2. (B) γ-30 lysates were immunoprecipitated for either A10 or NPR A as a control. Resulting immunoprecipitates were probed for the coIP of PS1 CTF. (C) Lysates from cells that express A10 only, human γ-secretase only, neither, or both (lanes 1, 2, 3, and 4, respectively) were specifically pooled to form mix 1 (A10 only + γ-secretase only) or mix 2 (neither + both) and then immunoprecipitated for γ-secretase with an M2 resin targeting the Flag tag on Pen-2. Immunoprecipitates were probed for the coIP of A10 and the γ components PS1 NTF and Pen-2. (D) γ-30 cells were treated with a nonpermeable biotinylation reagent at 4°C to label cell surface proteins. Lysates were immunoprecipitated for Aph-1, and the resultant immunoprecipitates were eluted and pulled down with streptavidin to enrich for cell surface interactors of γ-secretase, followed by blotting for α- and γ-secretases as in A. im, immature; m, mature.

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