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. 2007 May 2;104(19):8041–8046. doi: 10.1073/pnas.0611496104

Fig. 6.

Fig. 6.

Cryopyrin self-association and association with ASC require ATP binding. (A) Extracts from HEK293 cells transfected with plasmids encoding FLAG-tagged WT (Upper) or Walker A mutant (Lower) versions of cryopyrin were subjected to size-exclusion chromatography (as detailed in Materials and Methods). The chromatograms show UV absorbance plotted against volume of elution. Arrows indicate volume of elution for standards of the indicated molecular mass. Cryopyrin content (shown below the chromatographs) is determined by immunoblot analysis with anti-FLAG antibodies. (B) HEK293 cells were transfected with indicated plasmids. Lysates from the cells were subjected to immunoprecipitation with anti-HA antibodies (Left) and anti-FLAG antibodies (Right), and samples were analyzed by immunoblot analysis with the indicated antibodies. (C) THP1 cells were transduced with recombinant retrovirus expressing the following proteins: empty vector, no cryopyrin; FLAG-tagged cryopyrin; or Walker A mutant (WA) FLAG-tagged cryopyrin. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and analyzed by immunoblot with anti-FLAG and anti-ASC. The second and fourth lanes in Left have no sample.