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TAP-Bcl-xL is able to protect from apoptosis, localise correctly and co-purify with several cellular proteins.

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posted on 2010-12-20, 00:28 authored by Maria Teresa Vento, Valeria Zazzu, Alessia Loffreda, Justin R. Cross, Julian Downward, Maria Patrizia Stoppelli, Ingram Iaccarino

(A) HeLa cells stably expressing TAP-tagged Bcl-xL are more resistant than control to UV-induced apoptosis. Analysis of PARP cleavage in HeLa cells expressing TAP alone or TAP-Bcl-xL and irradiated or not with UV (200 J/m2). Cells were collected 2 and 4 hours after irradiation and analysed by immunoblot for the appearance of the cleaved product of PARP. (B) TAP-tagged Bcl-xL localises to the same intracellular compartment of endogenous Bcl-xL. HeLa cells expressing TAP-Bcl-xL were fractionated by differential centrifugation to obtain: lane 1, cytosolic proteins (S100); lane 2, nuclear proteins (Nuclei); lane 3, the heavy membrane fraction (HMM); lane 4, the light membrane fraction (LMM). 20 µg of each fraction were analysed by immunoblot for the presence of TAP-BclxL (using secondary antibody only) and endogenous Bcl-xL. The quality of the fractionation was determined using the antibodies against Sec23 (LMM), Cytochrome c (HMM), Bax (S100 and HMM) and PARP (Nuclei). (C) HeLa cells stably transfected with TAP alone or TAP-Bcl-xL were subjected to Tandem Affinity Purification. The figure shows a representative Coomassie stained SDS-PAGE performed under reducing conditions used to resolve eluates from purifications. Purifications were performed using membrane CHAPS extracts from HeLa expressing TAP alone (lane 1) or HeLa expressing TAP-Bcl-xL (lane 2).

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