Skip to main content
. 2018 Sep 4;9(69):33202–33214. doi: 10.18632/oncotarget.26059

Figure 6. Identification of non AUG initiation codons in SERPINC1.

Figure 6

(A) Squematic representation of the mutated pCEP4-S169A-M1I plasmid used in the recombinant model showing residues mutated to identify alternative initiation codons. Triangles represent stop codons in the reading frame of SERPINC1. (B) Antithrombin released to conditioned media (AT) of cells transfected with the pCEP4-S169A-M1I plasmid containing different mutations: missense or nonsense mutations affecting residues located before the original initiation codon (L-5V, L-5X; G-4X; T-1X); missense or nonsense mutations at or just after the original initiation codon (I1S, I1X, N4X); nonsense mutation affecting the first downstream AUG codons (M49X, M52X); and a nonsense mutation downstream of all potential initiation codons (R161X). (C) Intracellular expression of beta-actin as loading control for selected mutants, including those with no antithrombin released to the conditioned media (M52X and R161X). (D) Consequence on antithrombin from conditioned media of mutations affecting Ile1 in the pCEP4-S169A-M1I plasmid. (E) Effect on antithrombin from conditioned media of possible nonsense mutations affecting position 1 in the pCEP4-S169A-M1I plasmid. (F) Squematic representation of the wild-type pCEP4-S169A plasmid showing the mutation that severely disturbs the Kozak sequence (T-1X) in a wild-type context (Met1). Antithrombin from conditioned media and cell lysate as well as intracellular beta-actin were detected by Western blot. Full length wild-type antithrombin (58 KDa) is pointed by an arrow while small antithrombins (46 KDa) are pointed by grey arrows. Five-fold conditioned media of mutants was loaded compared to the wild-type.