Sirt6

Cells were collected and underwent lysis using a buffer (cat. no. AR0103-100; Boster Biological Technology). Protein was quantified using a BCA assay and protein lysates (40 µg) were then separated by 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes. The membranes then underwent 1 h of blocking in TBS (pH 7.6) containing 5% non-fat milk and 0.05% Tween 20 at room temperature. The membranes were incubated with primary antibodies overnight at 4°C and with the secondary antibodies at room temperature for 2 h. TGF-β (1:500; cat. no. sc-146), B-cell lymphoma-2 (Bcl-2; 1:500; cat. no. sc-7382), NF-κB P65 (1:500; cat. no. sc-7151), cleaved (cle)-PARP (1:500; cat. no. sc-56196) and Bcl-2-associated X (Bax; 1:500; cat. no. sc-7480) antibodies were purchased from Santa Cruz Biotechnology, Inc. SIRT6 (1:1,000; cat. no. ab191385), COL-1 (1:1,000; cat. no. ab34710), TNF-α (1:1,000; cat. no. ab6671), 3-NT (1:1,000; cat. no. ab61392) and GAPDH (1:1,000; cat. no. ab8245) antibodies were provided by Abcam. The primary and secondary antibodies (1:1,000; Cell Signaling Technology, Inc.; cat. nos. 7076 and 7074) were diluted in TBS-0.05% Tween-20 prior to use. The enhanced chemiluminescence kit (Bio-Rad Laboratories, Inc.) was used to detect the specific bands. Densitometric analysis was performed using Image Lab 5.1 software (Bio-Rad Laboratories, Inc.).