Western blotting (WB) analysis

The RIPA Lysis and Extraction Buffer (#89900, ThermoFisher) was used in the extraction of the PDAC cells total protein. The protein samples were next quantified by the bicinchoninic acid (BCA) Protein Assay Kit (#SB-WB013, ShareBio). An equal amount of the proteins within the lysates were then separated by the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then rapidly transferred onto the nitrocellulose membranes. After that, the membranes with protein bands were blocked within a 5% skim milk, cut, and incubated separately with target-specific primary antibodies relatively at 4 °C at 60 rpm overnight. The next day, the membranes were correspondingly incubated with species-specific secondary antibodies for an hour at room temperature. The immunoreactions were detected by a ChemiDoc XRS + chemiluminescence fluorescence imaging system (#1708265, BIO-RAD).

The primary antibodies involved in our study included anti-mTOR (1:1000, #2983, Cell Signaling Technology), anti-Phospho-mTOR (Ser2481) (1:1000, #2974, Cell Signaling Technology), anti-p70 S6 Kinase (1:1000, #9202, Cell Signaling Technology), anti-Phospho-p70 S6 Kinase (Thr421/Ser424) (1:1000, #9204, Cell Signaling Technology), anti-LIN28B (1:1000, #16178-1-AP, Proteintech), and anti-β-actin (1:2000, #4970, Cell Signaling Technology).

The only secondary antibody in this assay was the goat anti-rabbit IgG, HRP-linked antibody (1:5000, #7074, Cell Signaling Technology).