Image station 4000s pro

Cells were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich) or boiled directly in Laemmli’s buffer. Proteins were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. After blocking with 5% skim milk in PBST, membranes were incubated with primary antibodies overnight (Cell Signaling Technology; anti-α-tubulin #3873 at 1:2000, anti-β-actin #3700 at 1:2000 and anti-p53 #2524 at 1:1000 dilutions.) followed by HRP-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 h at room temperature. Detection was by ECL Plus (PerkinElmer) and a Kodak Image Station 4000s pro. Quantification was performed by densitometry using ImageJ software.

For Western blot analysis of cell culture medium, cells were seeded in six-well plates at 5 × 10⁵ cells/well and cultured for 24 hr in medium containing 10% FCS. The medium containing 10% FCS was removed and replaced by serum-free medium overnight. The medium was recovered and filtered using an Amicon Ultra-2 Centrifugal Filter (Merck Millipore) and boiled in Laemmli sample buffer for 5 min. Samples were then electrophoresed at 200 V on 12% SDS-polyacrylamide gels for 45 min, transferred to nitrocellulose membranes for 1 hr at 350 mA, blocked with 5% skim milk in PBS containing 0.05% Tween-20 (PBST) for 1 hr and incubated with anti-MMP1 (ab52631, Abcam), anti-MMP3 (ab53015, Abcam) or anti-MMP9 (ab76003, Abcam) overnight at 4ºC. Membranes were stained with horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature and then developed using Westar ETA C 2.0 (Cyanagen). Imaging was performed using a Kodak image station 4000s pro. Protein expression was quantified by densitometry using ImageJ software.