Tbs tween 20

Twenty to forty micrograms of proteins from whole extracts were loaded in a polyacrylamide gel (Bio Rad Laboratories, Hercules, CA, United States), separated by SDS–PAGE, and transferred on nitrocellulose membrane (Sigma-Aldrich, St. Louis, MO, United States) using Mini Trans-Blot BioRad (Bio Rad Laboratories, Hercules, CA, United States). nitrocellulose membranes were blocked with no-fat milk 5% w/v and incubate at +4°C overnight with specific primary antibodies as described in the section “Antibodies and chemical reagents.” The day after nitrocellulose membranes were washed three times per 5 min with TBS Tween-20 (Thermo Fisher Scientific, Waltham, MA, United States) and incubate at room temperature for 1 h with goat anti-rabbit or anti-mouse antibodies conjugated with HRP, used as a detection system (ECL) according to the manufacturer’s instructions (Amersham Biosciences, United Kingdom).

Typically, 15 to 30 μg of total cellular protein were loaded in polyacrylamide gel (Bio Rad Laboratories) and separated by SDS-PAGE. Thereafter, proteins were transferred onto nitrocellulose membrane (Sigma-Aldrich) using Mini Trans-Blot BioRad (Bio Rad Laboratories). Before to be incubated overnight at 4 °C with specific primary antibodies (MMP-2 and mMP-9, Invitrogen, 436000; MA5-15886 Waltham, MA, USA), membranes were blocked in no-fat milk (5% w/v) for 1 h. The day after, Horseradish Peroxidase (HRP) goat anti-rabbit or anti-mouse antibodies were added to the membranes for 1 h at room temperature. TBS Tween-20 (Thermo Fisher Scientific) was used to wash three times the membranes before and after each incubation procedure. Enhanced ChemiLuminescence (ECL) (Euroclone) was employed to detect HRP secondary antibodies signal. To conclude, protein bands were detected with Chemi Doc XRS (Bio-Rad, Hercules, CA, USA).

Cells were resuspended in 3–5 volumes of RIPA buffer, containing NP-40 (1%), sodium deoxycholate (0.5%), sodium dodecyl sulfate (SDS) (0.1%), aprotinin (10 μg/ml), leupeptin (1 mM), and PMSF (1 mM) and incubated on ice for 1 h. After centrifugation (18,000 × g for 15 min at 4°C), supernatant was collected and protein concentration was determined by Bradford method. Laemmli buffer 4 × was added to each sample before boiling at 95°C for 5 min. Typically, a quantity of 20–40 μg of total extracts was applied to polyacrylamide gel (Bio Rad Laboratories); thereafter, proteins were divided by weight in SDS–polyacrylamide gel electrophoresis (PAGE) and moved on nitrocellulose membrane (Sigma-Aldrich) using Mini Trans-Blot BioRad (Bio Rad Laboratories). In order to fill uncovered spots, obtained films were incubated in nonfat milk (5% w/v) and then blotted overnight with primary antibody according to the experimental procedures. The next day, HRP-conjugated goat anti-rabbit or anti-mouse was used to detect protein–antibody complexes. Each incubation was preceded and followed by 5 min wash with TBS Tween-20 (Thermo Fisher Scientific) for three times. Finally, nitrocellulose membranes were detected by standard chemical luminescence method ECL (Euroclone). Immunoblotting signals were captured using Chemi-Doc XRS (Bio-Rad Laboratories) and quantitatively analyzed by ImageJ (NIH, Bethesda).