Hybond enhanced

The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 1 mM EDTA, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 1 mM NaF, protease inhibitor cocktail). Cell lysates were separated to SDS-PAGE (8–15%) by electrophoresis and transferred onto a Hybond enhanced chemiluminescence (ECL) transfer membrane (Amersham Pharmacia, Piscataway, NJ, USA). After blocking in 3–5% skim milk, the membranes were probed with multiple antibodies against c-Myc (1:2000), RPL5 (1:1000), RPL11 (1:1000) (Abcam, Cambridge, United Kingdom), phospho-AKT (Ser473) (1:1000), AKT (1:1000), PARP(1:1000), Bcl-2 (1:1000), Bcl-x_L (1:1000), cyclin D1 (1:1000), CDK4 (1:1000), p53 (DO-1) (1:2000), MDM2 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) (1:500), p21 (Ab frontier, Seoul, Korea) (1:500), and β-actin (Sigma Aldrich Co., St. Louis, MO, USA) (1:3000) and exposed to horseradish peroxidase (HRP)-conjugated secondary anti-mouse or rabbit antibodies (1:5000) for 2 h. Protein expression was measured using the ECL system (Amersham Pharmacia, Piscataway, NJ, USA). Immunoprecipitation was performed using RPL5 or RPL11 and MDM2 antibodies. Then, protein G or A beads (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) were washed three times with lysis buffer. The final precipitated proteins were subjected to immunoblotting.

Following denaturation for 10 min at 65 °C, protein samples were separated by sodium dodecyl sulphate (SDS) 9% polyacrylamide gel electrophoresis (PAGE), and electroblotted onto a Hybond enhanced chemiluminescence (ECL) membrane (Amersham Biosciences). Antibodies for Collagen Type XII, Collagen Type XIV, Collagen Type XI alpha 2, Fibronectin and Prolargin were used. Further details on blocking solutions, primary and secondary antibodies used, as well as their respective host species, working dilutions and commercial suppliers can be found in Supplementary Table 2.
After ECL detection (Amersham Biosciences), bands were quantified using Quantity One 4.6.8 Software (Bio-Rad) and values were normalized to the total protein loading (density value of each complete lane, obtained after staining of the membrane following immunodetection with Page Blue Protein Staining Solution (ThermoFisher Scientific), using a protocol adapted from Welinder and Ekblad^{28 (link)}. All samples were run in the same SDS-PAGE gel, and background signal was measured in several different areas of the membrane. Average background signal was then subtracted to the band intensity signal to minimize background variation and interference.