Rabbit anti p16

Immunoblotting was performed using the following antibodies: rabbit anti-Bmi-1 (#5856; Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit anti-p16 (sc1207; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rat anti-p19Arf (sc32748; Santa Cruz Biotechnology, Inc.), rabbit anti-p15 (sc613; Santa Cruz Biotechnology, Inc.), rabbit anti-p18 (sc865; Santa Cruz Biotechnology, Inc.), rabbit anti-phospho-Rb (ser807/811) (#9308; Cell Signaling Technology, Inc.), rabbit anti-phospho-Akt (ser473) (#4060; Cell Signaling Technology, Inc.), rabbit anti-Akt (#4691; Cell Signaling Technology, Inc.), and mouse anti-β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA).

Snap frozen tissues from Ercc1^−/Δ and WT mice were lysed in RIPA lysis and extraction buffer (Thermo Fisher). Protein concentration was determined using BCA protein assay kit (Thermo Fisher). Equal amounts of protein were loaded onto SDS‐PAGE polyacrylamide gels then transferred to 0.2 µm pore size nitrocellulose membranes (Bio‐Rad). Membranes were blocked with 5% BSA in PBS‐Tween for 1 h at room temperature then incubated with primary antibodies overnight at 4℃. Membranes were then probed with secondary antibodies at room temperature. Protein expression was measured by fluorescence using iBright™ FL1000 Imaging System. The density of each blot was quantified by using ImageJ (NIH) normalized to GAPDH. The following primary antibodies were used in this study: rabbit anti‐GAPDH (Cell Signaling Technology, 2118), rabbit anti‐p16 (Santa Cruz, sc‐1207), rabbit anti‐p21 (Abcam, ab7960), rabbit anti‐p‐p65 (Cell Signaling Technology, 3033), mouse anti‐p‐p65 (Cell Signaling Technology, 6956), rabbit anti‐Pai‐1 (Santa Cruz, sc‐8979), rabbit anti‐COX2 (Cell Signaling Technology, 12282). The following secondary antibodies were used in this study: goat anti‐rabbit IgG (H+L) Alexa Fluor Plus 488 (Thermo Fisher, A32731), goat anti‐mouse IgG (H+L) Alexa Fluor 633 (Thermo Fisher, A21052).

Antibodies used were rabbit anti‐p21 (Cell Signaling Technology, 2947), rabbit anti‐p16 (Santa Cruz Biotechnology, sc‐468), mouse anti‐β‐actin (Millipore, Billerica, MAB1501), mouse anti–α smooth muscle actin (Sigma‐Aldrich, A2547), goat anti‐type I collagen (SouthernBiotech, 1310‐01), rabbit anti‐β‐catenin (Cell Signaling Technology, 8480), rabbit anti‐non‐phospho (active) β‐catenin (Cell Signaling Technology, 8814), rabbit anti‐histone H3 (Cell Signaling Technology, 4499), mouse anti‐CD81 (Santa Cruz Biotechnology, 555675), mouse anti‐CD9 (Santa Cruz Biotechnology, sc‐59140), mouse anti‐CD63 (BD Pharmingen, 556019), rabbit anti‐Hsc70 antibody (Proteintech, 10654‐1‐AP) and mouse anti–Tom20 (Santa Cruz Biotechnology, sc‐17764). Hoechst 33242 (Sigma‐Aldrich, H342) and collagen type I solution from rat tail (Sigma‐Aldrich, C3867) were purchased reagents.