FIGURE 4.

Truncated sema3A is a stable protein. One µg of purified P- and T-sema3A were incubated in 100 µL elution buffer in a 96-well plate at 37°C for 0-72 h. At the indicated time point, proteins were collected, and a sample buffer (without Dithiothreitol redox reagents) was added. All the samples were loaded on SDS-PAGE gel, then the membrane was immunoblotted with anti-human sema3A polyclonal antibody directed against the N-terminal (26-771 amino acids), followed by anti-goat HRP antibody. The bound antibodies were visualized using the EZ-ECL method and the blots were viewed by ImageQuant LAS 4000 machine and analyzed using ImageQuant TL Analysis software. The blue arrow points to the T-sema3A dimer (∼140 kDa), while the red arrow points to T-sema3A monomer (∼68 kDa), and the green arrow points to the P-sema3A dimer (∼160 kDa).