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. Author manuscript; available in PMC: 2013 May 9.
Published in final edited form as: Structure. 2012 Apr 5;20(5):887–898. doi: 10.1016/j.str.2012.03.001

Figure 6. Characterization of the Brox:CHMP5 and Brox:CHMP4B interfaces in the cell.

Figure 6

(A) Identification of Brox (left panel) and CHMP5 (right panel) residues important for the Brox:CHMP5 interaction in the cell. FLAG-tagged CHMP5 proteins (WT or the indicated mutant) were expressed in HEK293T cells in the presence or absence of HA-tagged Brox (WT or the indicated mutant). Forty-eight hours post transfection, cells were lysed in RIPA buffer and cleared lysates were incubated with anti-HA antibody conjugated beads. Both input and immunoprecipitated complexes were analyzed by western blot using the indicated antibodies.

(B) Fluorescence polarization assay for the Brox (left panel) and CHMP5 (right panel) mutants. The binding isotherms and the dissociation constants are marked and color-coded.

(C) Identification of Brox (left panel) and CHMP4B (right panel) residues important for the Brox:CHMP4B interaction in the cell. FLAG-tagged CHMP4B proteins (WT or the indicated mutant) were expressed in HEK293T cells in the presence or absence of HA-tagged Brox (WT or the indicated mutant). Examination of the protein interactions was performed similar to (A).

(D) Fluorescence polarization assay for the Brox (left panel) and CHMP4B (right panel) mutants. The binding isotherms and the dissociation constants are marked and color-coded.

See also Figure S5.