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.