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. 2023 Jan 10;13:1085892. doi: 10.3389/fphar.2022.1085892

FIGURE 1.

FIGURE 1

Generation of a modified truncated sema3A (A). The sequence of truncated sema3A (lower scheme) is a modified construct of the original/parental sema3A (upper scheme). The new construct truncated at amino acid 516 of the original sema3A with a point mutation at position 257, which exchanged a serine into a cysteine residue (S257C mutation), with 8xHis epitope tags at the C terminal. (B). The culturing medium of HEK293 cells expressing either P-sema3A or T-sema3A was collected and purified on a nickel affinity column to produce purified parental (P)-sema3A and truncated (T)-sema3A. The purified proteins were loaded on SDS-PAGE in the presence of Dithiothreitol reducing agent (+) or without (-). 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 bands in the blot represent the following: 1a is P-sema3A monomer (∼95 kDa), 2a and 4b are T-sema3A monomers (∼68 kDa). While 4a double band is T-sema3A homodimer (∼140 kDa) and 3a is P-sema3A dimer (∼160 kDa). Bands 1b and 3b are cleavage fragments of P-sema3A by furin protease.