Akt phosphorylation assays

The cell-based mechanistic studies of the fusion protein were carried out in HepG2 cells, which express extremely low levels of hepatic metabolism enzymes and altered TfR recycling mechanism^15,16, and are ineffective at converting ProINS-Tf to its active form¹⁷. Therefore, the prodrug form of the fusion protein, ProINS-Tf, is inactive in Akt signaling in this cell line.

Confluent HepG2 cells were starved in serum-free medium for 16–18 h before the experiment, and starved cells were then treated with different proteins for the indicated period of time. After treatment, cells were washed and lysed with cell lysis buffer (Cell Signaling Technology) supplemented with protease and phosphatases inhibitor cocktail (Thermo Scientific) on ice. After BCA quantification, equal amounts of total cellular proteins from each treatment group were subjected to Western blot analysis using anti-phospho-Akt antibody (Ser473, Cell Signaling) and anti-GAPDH antibody (D16H11, Cell Signaling). Band densities were quantified by Image Lab software (Bio-Rad, Hercules, CA).

For pulse-chase Akt phosphorylation assays, starved HepG2 cells were first pulsed with dosing medium containing different proteins for 30 min at 4 °C. Cells were then washed 3 times with ice-cold PBS and chased by incubation in protein-free DMEM medium at 37 °C for the indicated period of time.