Fig. 4. Double indirect immunofluorescence analysis of HeLa cells expressing both wild-type (A,E) and mutant forms of lamin C (B-D and F-H). In each case the cells were labeled with the 12CA5 anti-HA antibody to reveal the HA-tagged lamin C alleles (A-D) and a rabbit antibody against lamin A to reveal the endogenous protein (E-H). Each of the mutants shown caused the relocation of endogenous lamin A to the nucleoplasm and often induced the formation of lamin A-containing aggregates (arrows). This lamin A aggregation phenotype is most pronounced with LaC N195K (C and G). Higher-magnification confocal views of lamin A aggregates (J) resulting from LaC N195K overexpression (I) are also shown. Nucleoli (identifiable by phase contrast microscopy, not shown), which are unlabeled by either antibody, are indicated by arrowheads (I and J).