Westar eta c 2

Membrane proteins from heat-treated and control cells were extracted as described previously 49 (link) and protein concentration measured by Bradford assay (BioRad Laboratories Inc., Hercules, California, USA). Western blot analysis was conducted as reported previously 49 (link), using a mouse monoclonal NIS-specific antibody (Merck Millipore; dilution 1: 1700) overnight at 4 °C and a horse-radish peroxidase- labeled goat anti-mouse antibody (Jackson ImmunoResearch Europe Ltd., Ely, UK; dilution 1:2000) for 1 h at room temperature. After 1 min incubation with enhanced chemiluminescence Western blotting detection reagent (WESTAR ETA C 2.0; Cyanagen Srl, Bologna, Italy), images were taken with an ECL ChemoCam Imager (INTAS, Göttingen, Germany). As control for protein loading, the membrane was stripped (Restore Western Blot Stripping Buffer, Thermo Fisher Scientific) and re-probed with a monoclonal anti-β-actin antibody produced in mouse (Sigma Aldrich; dilution 1:1500). The intensity of the bands was measured by densitometry using ImageJ software (NIH, Bethesda, Maryland, USA) and normalized to the β-actin loading control, expressed as the relative amount of NIS protein.

One plate (35 mm dish) of NRK cells grown to ~80%–90% confluency were lysed with 0.6 mL of lysis buffer (2% SDS, 50 mM Tris-HCl (pH 6.8), 2 mM EDTA (pH 8.0), 1 mM PMSF (Sigma) and complete EDTA-free protease inhibitor cocktail (Roche)). The lysates were mixed with 4× loading buffer and heated at 95 °C for 10 min. Samples were separated by 10%/12%/15% SDS-PAGE and transferred onto nitrocellulose membrane (GE Company) or PVDF membrane (Millipore). The membranes were blocked in TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl and 0.1% Tween-20) containing 3% BSA at room temperature for 1 h. Primary antibodies were diluted in Solution I Buffer (TOYOBO). The membranes were incubated with primary antibodies at 4 °C overnight. After incubation, the membranes were washed three times with TBS-T and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in TBS-T containing 3% BSA at room temperature for 1 h. After final washes with TBS-T, the membranes were developed using Westar ETA C 2.0 (CYANAGEN) and exposure to X-ray film (Kodak).

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.