Ab7960

A549 cells and H1299 cells infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA were cultured for 2 days for protein isolation. In brief, cells were washed with PBS buffer and harvested with lysis buffer (100 mM Tris-HCl, pH = 7.4 l 0.15 M NaCl; 5 mM EDTA, pH = 8.0; 1% Triton X100; 5 mM DTT; 0.1 mM PMSF) to extract total proteins which were quantified with BCA Protein Assay Kit (Pierce, Rockford, IL, USA). To perform western blot analysis, 20 μg protein samples were mixed with loading buffer. Then SDS-PAGE electrophoresis and subsequent PVDF transmembrane were performed (Amersham Biosciences, Pollards Wood, UK). Membrane was blocked with 5% milk dissolved in TBST buffer for 1 h and then incubated with primary antibodies overnight at 4 °C. Primary antibodies used here were as follows: Rabbit anti-GMDS, Novus Biological, NBP1–33424 (1:500); Rabbit anti-CDKN1A, Abcam, ab7960 (1:500); Mouse anti-DDIT3, Abcam, ab11419 (1:1000); Rabbit anti-FAS, Abcam, ab82419 (1:1000); Rabbit anti-JUN, Abcam, ab32137 (1:1000); Rabbit anti-VEGFA, Abcam, ab183100 (1:500); mouse anti-Flag, Sigma, F1804 (1:1000); mouse anti-GAPDH, Santa-Cruz, sc-32,233 (1:2000). After washing with TBST buffer for three times, specific HRP conjugated secondary antibodies from Santa Cruz were added and immunoactivity was detected with ECL-Plus kit (Amersham Biosciences, Pollards Wood, UK).

Rabbit anti-FLAG polyclonal (Sigma F7425), rabbit anti-HA (haemagglutinin) polyclonal (Santa Cruz Y-11 [sc-805]), rabbit anti-p21 polyclonal (Abcam ab7960), mouse anti-Nac1 monoclonal (Abcam ab81987) and mouse anti-GAPDH monoclonal (Calbiochem CB1001) antibodies were used in western blots at 0.5, 2, 5, 0.82 and 1 μg/ml, respectively. Horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies (Pierce 31460 and 31430, respectively) were used at 40 ng/ml.

Cells were lysed for 30 min on ice in lysis buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethanesulphonyl-fluoride, 5 μg/ml aprotinin, 20 μg/ml leupeptin). Lysates were clarified by centrifugation, and twenty μg of protein extracts were fractionated by SDS-PAGE and transferred onto nitrocellulose using standard protocols. Equal protein loading was verified by Ponceau staining. Filters were blocked in PBS 1X Tween-20 with 5% of skim milk and incubated overnight with primary specific antibodies for α-SMA (A2547; Sigma-Aldrich), FAP (sc-65398; Santa Cruz Biotechnology), p21 (ab7960; Abcam), p16 (ab7962; Abcam) and Vinculin (V9131; Sigma-Aldrich). The filters were then incubated with the secondary peroxidase linked whole antibodies. Bound antibody was detected using the Novex ECL, HRP Chemiluminescent substrate Reagent Kit (Life Thecnologies).

Total protein extracts were prepared according to standard methods. Forty micrograms of protein extract was fractioned by SDS-PAGE and transferred onto Hybond nitrocellulose filters (RPN 303D, GE Healthcare, Milan, Italy). Filters were blocked in PBS-Tween 20 and 5% skim milk and incubated overnight with the following primary antibodies: mouse monoclonal anti-BCL-2 (sc-509, Santa Cruz Biotechnology, Dallas, TX, USA), anti-KIT (#3308; Cell Signaling Technology, Danvers, MT, USA), and anti-MYC (ab32; Abcam, Cambridge, United Kingdom); rabbit polyclonal anti-AKT(S473) (sc-9271, Santa Cruz Biotechnology), anti-BRAF (ab33899, Abcam,), anti-ERK1/2 (T202/Y204) (#9101, Cell Signaling Technology), anti-γ-H2AX (ab11174, Abcam), anti-p21^waf1 (ab7960, Abcam), and anti PARP-1 (#9542, Cell Signaling Technology). Mouse monoclonal anti-Vinculin (VCL, V9131, Sigma-Aldrich, Milan, Italy) or anti-β-Actin (Ab8226, Abcam) antibodies were used to ensure equal protein loading. The filters were then probed with secondary peroxidase-linked whole antibodies (GE Healthcare, Milan, Italy) and subjected to autoradiography using the Novex^® Enhanced Chemoluminescent Horseradish Peroxidase detection system (Thermo Fisher Scientific, Monza, Italy). Films were scanned (ImageScanner III, GE Healthcare, Milan, Italy) and images were processed by Photoshop7.0.1 or analyzed using ImageJ 1.46r.

Cell lysates were obtained using RIPA (20 mM Tris-HCl pH = 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). A total of 60–120 μg protein were fractionated by SDS–PAGE and transferred to PVDF membranes (GE Healthcare Life Sciences, Buckinghamshire, UK). These were blocked in 5% nonfat dry milk in TBS-T (20 mM Tris-HCl pH = 7, 5, 150 nM NaCl, 0,1% Tween20) for one hour and immunoblotted. Blots were developed using ECL Detection Reagent (GE Healthcare Life Sciences), images acquired on ImageQuant LAS 4000 (GE Healthcare Life Sciences) and analyzed using Image J 1.52p (National Institute of Health, Bethesda, MD, USA). Primary antibodies were used against HPV-18 E6 (AVC #1006, Arbor Vita Corporation, Fremont, CA, USA), p53 (sc-126, Santa Cruz, Dallas, TX, USA), pRb (ab24, abcam), p16INK4a (ab16123, abcam, Cambridge, UK), p21 (ab7960, abcam), PCNA (ab29, abcam, Cambridge, UK) and α-tubulin (T9026, Sigma-Aldrich, St. Louis, MO, USA).

P21, the protein encoded by the CDKN1A gene was determined using immunoblotting. 20 μg of protein was resolved by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis on 10% polyacrylamide gels. Proteins were transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA), blocked with 5% dry milk (Bio-Rad, München, Germany) in TBS (Sigma) overnight at 4°C and probed with primary antibodies primary antibody against p21 (ab 7960) (Abcam, Bristol, UK) during 1 h at room temperature. Proteins were then visualized using the ECL Detection System (Pierce, Rockford, IL) as per the manufacturer’s instructions.