3.7 Immunoblotting and chromosomal fusion analysis
To validate the wildtype and HGPS mutant lamin A transfection we
performed immunoblotting. Briefly protein lysate was prepared and
stained with lamin A antibodies, results for immunoblotting was shown in
the (Figure 4A). Results clearly shows that, in wildtype lamin A
transfected cells, single prominent band signal was visualized confirms
lamin A. Rather, protein samples from HGPS mutant lamin A transfection
cells, two closely related signal visualized and marked as red color
arrow in the (Figure 4A). The difference between mature lamin A and HGPS
mutant lamin A is only 39 amino acids. Hence, two close signals were
visualized in lane 3 and 4 of (Figure 4A). To confirm the data, we
analyzed the blot with gel analyzer software, which clearly shows the
presence of single band in wild type and double band in HGPS mutant
cells. Results for the gel analysis is shown in the (Figure 4B). Black
and Red asterisk indicates wild type and HGPS mutant lamin A,
respectively. Further to study the chromosomal fusions in HGPS mutant as
well as wild type lamin A transfected HDF cells, we performed metaphase
arrest after the transfection. Results for metaphase arrest for
chromosome fusion analysis was shown in the (Figure 4C, D). The data
clearly shows that, chromosomal fusion was noted in the HGPS mutant and
not in the wild type and control lamin A transfected cells. In addition,
it is clear that, HGPS mutation causes loss of 39 amino acid residues at
the C-terminal end of lamin A, which induce chromosomal fusion.