Sds polyacrylamide gel electrophoresis

Cells or lung samples were lysed in lysis buffer (Cell Signaling), briefly sonicated, incubated on ice for 30 minutes, and centrifuged at 12,000 g for 10 minutes. Supernatants were collected and stored at –80°C until use. For protein fractionation, 10 µg cell lysate was loaded per lane on gels for fractionation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Life Technologies), and transferred onto a nitrocellulose membrane. After overnight incubation with a primary antibody, the membrane was counter-stained with horseradish peroxidase-conjugated anti-rabbit or -mouse IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and visualized with an enhanced chemiluminescence system (Cell Signaling). Blots were photographed, digitized and analyzed using Image J, a public domain software developed by the NIH.

Transduced SKBR-3 and BT474 cells were cultured for 48 h and lysed using lysis buffer (Hepes pH 7.4 20 mM, NaCl 150 mM, 10% Glycerol, 1% Triton X-100) with protease inhibitors cocktail Complete (Roche Applied Science, Penzberg, Germany) and sodium orthovanadate or Phostop (Roche) used both as phosphatase inhibitors. Protein quantification was performed using Bradford protein assay (BioRad) and protein extracts were resolved on SDS-polyacrylamide gel electrophoresis (Invitrogen). Gels were then blotted onto nitrocellulose (GE Healthcare, Little Chalfont, UK) membranes and probed with appropriate primary antibodies (Table S3). Secondary antibodies were horseradish peroxidase-conjugated: anti-mouse or rabbit (Thermo Fisher Scientific) and anti-goat (Santa Cruz Biotechnology Inc.) and proteins detection was performed with ECL Detection Reagent (GE Healthcare) according to manufacturer's protocol. ECL signals were detected and measured by the Uvitec Chemiluminescence Imaging System and NineAlliance software (Uvitec Ltd., Cambridge, UK). ECL signals were detected and measured by the Uvitec Chemiluminescence Imaging System and ImageJ software (Bandyopadhyay et al., 2014 (link)).

Cells were lysed (lysis buffer: 20 mM HEPES pH7.5, 250 mM KCl, 5% glycerol, 10 mM MgCl₂, 0.5% Triton X-100, Protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Cell Signalling)) and sonicated. Lysates were cleared by centrifugation. Typically, 30 μg of protein lysate was separated on a 4–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Invitrogen) blocked in Odyssey buffer (LiCor Biosciences) and immunoblotted with the indicated antibodies. Immunoblots were imaged using an Odyssey infra-red imaging system (LiCor).

Isolation of total protein from IDD tissues or NP cells was performed with RIPA buffer. The concentration of protein was determined with the bicinchoninic acid (BCA) protocol. An equal amount of protein was resolved by SDS-polyacrylamide gel electrophoresis (Invitrogen, USA) and transferred to the PVDF membrane (Millipore). After blocking with 5% milk, the membrane was incubated with primary antibody (anti-MMP-9, No. MA5-15886; anti-ADAMTS-5, No. PA5-14350; Invitrogen). After washing three times with TBST, the membrane was incubated with an HRP-conjugated secondary antibody. The signals were generated with the chemiluminescent reagents. The primary antibodies used in this study are as follows: VEGF, MMP-9, ADAMTS-5, and GAPDH (Santa Cruz Biotechnology).

Monolayers of confluent cells were washed twice in PBS and then lysed in 1× Cell Lysis Buffer supplemented with 1 mM phenylmethylsulfonylfluoride (Cell Signaling Technology, MA). Fifty micrograms of total protein were subjected to SDS-polyacrylamide gel electrophoresis (Invitrogen, CA) for each cell line followed by immunoblotting with mouse monoclonal E-cadherin, N-cadherin and vimentin antibodies (BD Bioscience, NJ) and BAP1 antibody (Santa Cruz Biotechnology, CA) (1:1,000 in 5% blocking reagent in Tris-buffered saline/Tween-20) overnight at 4°C. The following day, blots were incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology; 1:1,000 dilution) for 1 hour at 25°C. Signals were visualized with enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, NJ) on X-ray film (Eastman Kodak, NY).