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. Author manuscript; available in PMC: 2017 Mar 9.
Published in final edited form as: Cell Host Microbe. 2016 Mar 9;19(3):336–348. doi: 10.1016/j.chom.2016.02.004

Figure 4.

Figure 4

NC binding to cellular membranes is necessary for the capture of ALIX Bro1. (A) NC zinc fingers are necessary for ALIX Bro1 capture. GST alone (lane 1), GST-NC-p6 (lane 2), GST-RKI or GST-RKII (lanes 3 and 4) were purified and subsequently tested for binding with HA-ALIX Bro1- containing cellular extracts. Captured complexes and input fractions were analyzed by SDS-PAGE and WB. GST proteins were visualized by Coomassie blue. (B) RNA removal ablates NC-ALIX Bro1 interactions. Pull down assays similar to those described in (A) were conducted with GST control (lane 1) and GST-NC (lane 2) without (lanes 1 and 2) or with nuclease treatment (lanes 3 and 4). Input and eluate fractions were analyzed as described above. (C) GST pull down assays were conducted in conditions similar to those described in (B) except in the way the HA-ALIX Bro1 extracts were prepared. 293T cells were either lyzed in presence of detergent (lanes 1–3) or in hypotonic buffer and lyzed mechanically (lanes 4–6) and interactions with NC-p6-ALIX Bro1 were assessed with or without nuclease. Input and eluate fractions were analyzed as described above.