Western Blot Protocol for Protein Analysis

The Western blot analysis was performed as reported previously^{14 (link)}. Total cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Buckinghamshire, UK). The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 1 h, and incubated with the primary antibodies listed next for 1 h with shaking. The primary antibodies against TG2, β-catenin, p-β-catenin, non-p-β-catenin, Ampka, p-Ampka, Foxo3a, p-Foxo3a, p65, p-p65, mTOR, p-mTOR, Axin2, Tcf1, Sirt1, and IκB were purchased from Cell Signaling Technology (Danvers, MA); antibodies against Sox9, Runx2, Mmp3, Mmp13, and Adamts5 were from Abcam (Cambridge, UK); the antibodies against LaminB and haemagglutinin (HA) were from Santa Cruz Biotechnology (Dallas, TX), and β-actin antibody was from Sigma Aldrich (St. Louis, MO). Membranes were washed, and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. The membrane signal was visualized using Supersignal Chemiluminescent Substrates (Thermo Fisher Scientific, Waltham, MA).