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. Author manuscript; available in PMC: 2014 Mar 26.
Published in final edited form as: Biochem J. 2013 Feb 1;449(3):613–621. doi: 10.1042/BJ20121198

Figure 3. Mutational analysis of ASC CARD surfaces with respect to foci formation with WT Caspase-1 CARD.

Figure 3

A. HEK293T cells were transiently transfected with VN-Caspase-1 CARD and VC-ASC CARD WT or ASC CARD mutants, respectively. 24 hours after transfection, foci formation was analyzed using fluorescence microscopy. Representative images from fluorescent microscopy are shown. ASC mutants R125D, E130R, D134R, Y137E, E144R, R160E, and D191R lead to diffuse phenotype, whereas mutants D134A, Y146E, Q145A, and R150E resulted in foci formation (F= Foci, D=diffuse staining). B. Immunostaining of total cell lysates showing proper expression of fusion proteins. Cell lysates were probed for ASC and for ß-actin as a loading control. C. Cells transfected with either WT or mutants (E130R, D134R, E144R, Q145A, Y137E) VC-ASC CARD and WT VN-Caspase-1 CARD were analyzed in the BiFC assay 24 h post transfection and then lysed by adding Native PAGE lysis buffer (Invitrogen) and sonication. Total lysates were applied to Native PAGE and immunoblotted for ASC and VN-GFP (depicting Caspase-1 CARD). Only the mutants causing a “diffuse” phenotype showed the presence of low order ASC-Caspase-1 CARD assemblies, which were able to enter the gel-matrix. Highly-oligomeric assemblies with very low mobility are shown above. Cell lysates were also probed for ß-actin as a loading control (bottom).