Clarity ecl chemiluminescence substrate

Cell lysates were obtained using RIPA buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1.0% Triton X-100, 0.1% SDS, 5 mM EDTA, pH 8.0) with a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Bradford assay was used to evaluate protein concentration in each sample. Proteins (25 μg) were analysed with SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Then, 5% skim milk was used to block the membrane, which was then incubated overnight with the primary antibody. Rabbit anti-phospho-NF-κB (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ACE2 (Cell Signaling), mouse anti-TMPRSS2 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) were used as primary antibodies. Horseradish peroxidase-conjugated antibodies anti-mouse or anti-rabbit (The Jackson Laboratory, Bar Harbor, ME, USA) were used as secondary antibodies. Protein bands were visualised by using the Clarity ECL chemiluminescence substrate (Bio-Rad) with Uvitec Imager (UVItec, Cambridge, UK) and then quantified using ImageJ software 4.1.

RIPA buffer (0.1% SDS, 150 mM NaCl, 1.0% Triton X-100, 10 mM Tris, 5 mM EDTA pH 8.0) with a protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) was used to obtain the cell lysates. The Bradford assay was used to evaluate the protein concentration in each sample. Proteins (25 μg) were analyzed by SDS-PAGE, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). A blocking buffer (Bio-Rad, Hercules, CA, USA) was used to block the membrane that was then incubated overnight with primary antibodies.
Mouse anti-phospho-p38 (Cell Signaling), rabbit anti-phospho-NF-kB (Cell Signaling), mouse anti-PCNA (Cell Signaling) and mouse anti-β-actin (Cell Signaling) were used as primary antibodies.
We used secondary horseradish peroxidase-conjugated antibodies (anti-mouse or anti-rabbit) (The Jackson laboratory, Bar Harbor, ME, USA). A Uvitec Imager (UVItec, Cambridge, UK) was used to visualize the protein bands by using the Clarity ECL chemiluminescence substrate (Bio-Rad) that were then quantified using ImageJ software. Each measure was normalized with respect to β-actin.

Cell lysates were obtained using RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 5 mM EDTA, pH 8.0) with protease inhibitor Cocktail (Roche Applied Science, Indianapolis, IN, USA). Protein concentration for each sample was evaluated by Bradford assay. Proteins (30 μg) were analysed by SDS-PAGE, and then transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). 5% skim milk was used to block the membrane that was then incubated overnight with the primary antibody.
Mouse anti-SIRT1 (Abcam), rabbit anti-Caspase-1 p10 (Santa Cruz Biotechnology), mouse anti-p16^ink4a (Santa Cruz) and, rabbit anti-α-tubulin (Cell Signaling), were used as primary antibodies.
Secondary horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from horse and goat respectively (Vector laboratories, CA, USA). Protein bands were visualized using Clarity ECL chemiluminescence substrate (Bio-Rad) with Uvitec Imager (UVItec,Cambridge,UK) and then quantified using ImageJ software.
Caspase 1 activity was measured as the ratio between band intensity of the pro-Caspasi-1 and of the cleaved Caspasi-1. Each measure was normalized with α-tubulin.