4.7. AKT and ERK Pathways Analysis

To verify the modulation of the MAPK and PI3K/AKT pathways by lysicamine, a Western blotting assay was performed. For this purpose, 2 × 10⁵ cells plated in 6-well plates for 24 h were treated with 0.5× IC₅₀, IC₅₀, or 1.5× IC₅₀ lysicamine or control in serum-starved media overnight. Next, the medium was removed, and a serum stimulus (10% FBS) with 0.5× IC₅₀, IC₅₀, or 1.5× IC₅₀ lysicamine concentration or control was added, and cells were incubated for 30 min.

The cells were harvested with ice-cold lysis buffer (10 mM NaCl, 50 mM Tris-HCl, 0.5% NP40, 0.1 mM EDTA, 0.1 M NaVO₄, complete protease inhibitor, Mini, Roche, 1×). After centrifugation (10,000 rpm, 5–10 min, 4 °C), the protein concentration was measured using the Qubit™ Protein and Protein Broad Range (BR) Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). The total protein extract (50 μg) was separated using a 10% polyacrylamide gel and transferred to a PVDF membrane. Membranes were blocked in TBS 5% skimmed milk solution for 30 min at room temperature. Furthermore, they were incubated for 16 h at 4 °C with appropriated primary antibodies, as described in the following: Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit (P-MAPK) (1:3000 dilution) and Phospho-Akt (Ser473) (D9E) XP^® Rabbit (dilution 1:1000) (Cell Signaling Technology, Danvers, MA, USA). All were diluted in TBS with 5% BSA. Next, membranes were incubated for 40 min with the following secondary antibodies: anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP) (dilution 1:5000) or anti-mouse conjugated to HRP (1:10,000) (Sigma-Aldrich St. Louis, MO, USA). Proteins were detected using SuperSignal™ West Atto Ultimate Sensitivity Substrate (ThermoFisher Scientific, Waltham, MA, USA) and visualized with the iBright CL750 Imaging System photodocumenter (ThermoFisher Scientific, Waltham, MA, USA).