Supersignal west pico plus

Cells were lysed in 100 μL 1X Laemmli buffer [62.5 mM Tris HCl pH 6.7, 10% glycerol, 2% sodium dodecylsulfate (SDS), 24 mM dithiotreitol (DTT), 2 mM Vanadate, bromophenol blue], heated at 90 °C for 5 min, and resolved by SDS-polyacrylamide gels electrophoresis, transferred to nitrocellulose membranes, and probed with primary antibodies. Protein signals were revealed by chemoluminescence (SuperSignal^TM West Pico PLUS, Thermo-Fisher Scientifics) and detected using a CCD camera (Fujifilm LAS-3000 Imager, Fuji, Tokyo, Japan). Quantification of Western blots signal intensities was done using the ImageJ software. Antibodies used are listed below (Table 2).

Cells and exosomes were lysed in radioimmunoprecipitation assay (RIPA) buffer (Pierce® 899000; Thermo Fisher Scientific) and protease cocktail inhibitor‐EDAT free (cOmplete Mini, #11836170001; Sigma–Aldrich). Protein concentration was determined by Pierc ™ BCA protein assay kit (#23225; Thermo Fisher Scientific). Approximately 20 µg protein was loaded on 10% SDS‐PAGE and then the separated proteins were blotted onto nitrocellulose membrane (Thermo Fisher Scientific). The membranes were washed three times with Tris buffered saline containing 0.1% of tween 20 (TBST) pH 6.5 for 10 min/washing step followed by masking with 5% BSA for 1 h. Membrane development was performed by incubating the primary antibodies (ALIX, CD9, CD81, CD63, flotillin‐1, TSG101, Grp94, promini‐2 and β‐actin) with the recommended dilution from suppliers overnight at 4°C. The membranes were washed three times (10 min each) with TBST and then the membranes were incubated with the corresponding secondary antibodies conjugated to HRP for 1 h at ambient temperature. Following washing steps with TBST, target proteins were detected using an enhanced chemiluminescence kit SuperSignal^Tm West Pico Plus (Thermoscientific, Rockford, USA).

Immunoblotting analysis was performed as previously described [37 (link)]. Briefly, tissues were homogenized on ice in RIPA buffer (Thermo Fisher Scientific) containing protease inhibitor cocktail (Roche, Basel, Switzerland). Homogenate was centrifuged at 4°C for 15 minutes at 12,000 rpm and the supernatant was used for protein concentration determination using BCA kit (Thermo Fisher Scientific). Equal amounts of total protein (30 μg) were reduced by adding Laemmli sample buffer containing dithiothreitol and heating to 90˚C for 5 minutes. Proteins in the samples were separated on 12.5% sodium dodecyl sulfate (SDS)- polyacrylamide gels, then transferred to nitrocellulose membranes, blocked in phosphate-buffered saline (PBS) containing 5% nonfat milk and 0.1% Tween-20 (PBS/T) and incubated overnight with ACE2 or TMPRSS2 antibodies at the dilutions indicated in S1 Table. After incubations with the secondary antibodies and chemiluminescence substrate (SuperSignal^TM West Pico PLUS, Thermo Scientific, Waltham, MA), protein bands were detected by autoradiography. Signals were quantified using densitometry with Image-J software (NIH, Bethesda, MD) and normalized to β-actin (ACTB) signals.

Lung tissues and cells were lysed in ice-cold RIPA lysis buffer containing protease and phosphatase inhibitors. The supernatants were collected by centrifugation and quantitated through BCA protein assay kit (Beyotime Institute of Biotechnology). An equivalent amount of 30 μg protein was separated by SDS-PAGE, transferred to a 0.22 μm PVDF membrane (Millipore Co, Bedford, MA, USA), and blocked in 5% BSA for 2 h at room temperature. The PVDF membrane was then incubated with the primary antibody overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies for 2 h in the dark. An ECL kit (Supersignal^TM West Pico PLUS, Thermo, USA) was used to detect the protein bands, and the grey level of proteins were calculated by the Image J software (National Institutes of Health, Bethesda, MD, USA). We diluted the antibodies as follows: rabbit anti-GAPDH (1:1000), rabbit anti-LC3B (1:1000), rabbit anti-P62 (1:1000), rabbit anti-ATG5 (1:1000), rabbit anti-NF-κB p65 (1:1000), rabbit anti-phospho-NF-κB p65 (1:1000), rabbit anti-ERK p65 (1:1000), rabbit anti-phospho-ERK (1:1000), rabbit anti-IRF 3 (1:1000), rabbit anti-phospho-IRF 3 (1:1000) and goat anti-rabbit IgG (1:4000). In the experiment, GAPDH was used as a protein loading control.

Western blotting was performed using a standard protocol established in our laboratory. Infected cells were harvested by the treatment of trypsin- EDTA (Life Technologies, Carlsbad, CA, USA) at different time points and were washed twice with PBS, then lysed in ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) with a protease inhibitor (ThermoFisher Scientific, Waltham, MA, USA) and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The total protein content of the extract was quantified using NanoDrop^TM 2000 (ThermoFisher Scientific, Waltham, MA, USA). Cell lysates (approximately 20 μg of protein) were loaded by SDS-PAGE and transferred into a nitrocellulose membrane (0.45 mm pore size, ThermoFisher Scientific, Waltham, MA, USA). The membrane was blocked using 0.05 g/mL blotting-grade milk powder (Bio-Rad, Hercules, CA, USA) for two hours, then incubated with primary antibody for overnight incubation on an orbital shaker. After overnight incubation, the antigen-antibody complex was visualized with HRP-conjugated goat anti-rabbit or anti-mouse IgG (Cell Signaling, Beverly, MA, USA), then developed with an ECL detection system (Supersignal^TM West Pico PLUS, ThermoFisher Scientific, Waltham, MA, USA) using the Bio-Rad ChemiDoc imaging system.

Cells resuspended in cell lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 10 mM EDTA, 0.1% SDS) were lysed by sonication. Protein lysates were cleared by centrifugation and 40 µg of total protein were separated on a 10% SDS-polyacrylamide gel. After semi-dry blotting to a nitrocellulose membrane (Carl Roth, Darmstadt, Germany), immunodetection was carried out with primary antibodies against PARP-1 (Cell Signaling Technology, Danvers, MA, USA; #9542), p53 (abcam, Cambridge, UK; ab26) and β-actin (Sigma-Aldrich, Taufkirchen, Germany; A2066), followed by incubation with respective secondary antibodies coupled to horseradish peroxidase (HRP). The SuperSignal^TM West Pico Plus (Thermo Fisher Scientific, Waltham, MA, USA) chemiluminescent substrate was added and signals were recorded with a Fusion Solo S imaging system (Vilber Lourmat, Eberhardzell, Germany).