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Figure 6


Fig. 6. FLAG affinity chromatography of FLAG-tagged Apc7 and Apc8 complexes. (A) Western blots of total protein extracts from S2 cells transfected with HA-tagged Apc8 (H8, lane 1) or FLAG-tagged Apc7 (F7, lane 2) plasmids. Strip 1 was reacted with anti-HA ({alpha}H, lane 1), and strip 2 with anti-FLAG ({alpha}F, lane 2) monoclonal antibodies. (B) Western blots of proteins affinity-purified on anti-FLAG-M2 antibody beads from S2 cells co-transfected with FLAG-Apc7 and HA-Apc8 (F7-H8, lanes 3 and 4), FLAG-Apc8 and HA-Apc7 (F8-H7, lanes 5 and 6) or FLAG-Apc7 and HA-Apc3 (F7-H3, lanes 7 and 8) plasmids. Blots were reacted with anti-FLAG (lanes 3, 5 and 7) or anti-HA (lanes 4, 6 and 8) monoclonal antibodies. The immunoreactive bands labelled with an asterisk are proteolytic degradation products of Apc8. (C) Blots of anti-FLAG-M2 column-bound (lane 10) and unbound (flow-through, lane 9) proteins from S2 cells transfected with HA-Apc8 (H8) plasmid and incubated with anti-HA monoclonal antibody. Even after heavily overloading the anti-FLAG column, no nonspecific binding of HA-Apc8 could be detected. Similarly, HA-Apc3 did not bind to the anti-FLAG-M2 column (data not shown) E, eluate; FT, flow-through.





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