Irdye 680rd or 800cw

Denatured proteins were resolved using either 4–12% Bis-Tris gels or 10–20% tricine gels (ThermoFisher) according to manufacturer’s recommendations. Proteins resolved on Bis-Tris gels and tricine gels were transferred respectively onto 0.45 μm and 0.22 μm nitrocellulose membranes (ThermoFisher) and probed with appropriate primary antibodies (Additional file 1: Table S3). Secondary antibodies conjugated with IRDye® 800CW or 680RD (LI-COR Biosciences) were used and images captured with the Odyssey® infrared imaging system (LI-COR Biosciences). For densitometric quantification, the intensity of the bands was determined using Image Studio™ Lite software (LI-COR Biosciences).

Experiments using primary cells were conducted using the following numbers of cells: 4 × 10⁸ human platelets per immunoprecipitation and 1 × 10⁷ per lane of whole cell lysate; 1.6 × 10⁸ mouse platelets per immunoprecipitation and 4 × 10⁶ per whole cell lysate; and 2.2 × 10⁶ HUVECs per immunoprecipitation and 5.5 × 10⁴ per whole cell lysate. Whole cell protein lysates and co-immunoprecipitation experiments were performed as previously described (8 (link)). Briefly, cells were lysed in 1% digitonin lysis buffer (10 mm Tris, pH 7.4, 150 mm NaCl, 0.02% NaN₃). Proteins were immunoprecipitated with primary antibody (as indicated in text) bound to protein G-Sepharose beads for 90 min and washed in 0.1% digitonin lysis buffer. Standard protocols were used for Western blotting and SDS-PAGE. Primary antibodies were used as indicated in the text with corresponding horseradish peroxide (Pierce) or IRDye® 680RD or 800CW (LI-COR Biosciences)-conjugated secondary antibodies. Membranes were visualized using Pierce ECL Western blot substrate (Thermo Scientific) and exposure to film or using an Odyssey Infrared Imager (LI-COR Biosciences). All quantitation was performed using an Odyssey Infrared Imager (LI-COR Biosciences); background signal was removed, and individual band intensities were compared.

Proteins were extracted to Cell Lysis Buffer (9803S; Cell Signaling Technologies) with Halt™ Protease Inhibitor cocktail and phosphatase inhibitors (Thermo Fisher Scientific). The protein concentrations measured with DC protein assay (Bio-Rad Laboratories). Proteins (30 µg) in the sample loading buffer (8 M urea, 2% SDS, bromophenol blue) were separated by electrophoresis in reducing conditions on a 12% sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE) with PageRuler Plus Prestained protein ladder (Thermo Fisher Scientific; #26620) and transferred onto Immobilon^®-FL PVDF membrane (Millipore). After blocking the membrane with Odyssey^® blocking buffer (LI-COR, Lincoln, NE, USA), 1 µg/ml anti-KLF2 antibody (Thermo Fisher Scientific; #PA5-40591) or 0.5 µg/ml mouse anti-GAPDH (Thermo Fisher Scientific; #437000) antibodies were allowed to bind o/n at 4°C or 2h at RT correspondingly. After incubation with 1:10 000 anti-mouse or anti-rabbit secondary antibodies (IRDye 680RD or 800CW) (LI-COR Biosciences) for one hour at room temperature (RT), the immune complexes were detected by Odyssey infrared scanner (LI-COR Biosciences). The band intensities were analyzed by Image Studio Lite Ver 5.2 from three individual analyses (LI-COR Biosciences).

30 µg proteins in sample buffer (8 M urea, 2% SDS, bromophenol blue) were separated on an 8 % or 12% SDS-PAGE gel and transferred onto Immobilon-P membrane (Millipore). Non-specific binding was blocked with Odyssey® blocking buffer (LI-COR, Lincoln, NE, USA). The membrane was incubated with 2 µg/mL polyclonal FXYD5 antibody (Acris Origene, Herford, Germany), 25 ng/mL monoclonal anti-beta catenin antibody (E247, Abcam), 250 ng/mL monoclonal anti-E-Cadherin antibody (BD Biosciences, San Jose, CA, USA), 25 ng/mL monoclonal anti-β-actin (Abcam) overnight at 4 °C and washed 3×5 min with Tris-buffered saline with 0.1% Tween-20 (TBS-T). After incubating the membrane in anti-mouse or anti-rabbit secondary antibody (IRDye 680RD or 800CW, LI-COR, 1:10 000) and washing as above, the fluorescence was recorded on Odyssey infrared scanner (LI-COR).

Protein was extracted with radio-immuno precipitation assay (RIPA) buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and a protease inhibitor cocktail. 10mg of total protein was separated on SDS-PAGE and then transferred to PVDF membranes using the Bio-Rad Trans-Blot protein transfer system. The membrane was blocked at room temperature for 90 min with 5% skim milk in 1 × TBST (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20). After blocking, the membrane was incubated with SARS-CoV-2 nucleocapsid antibody (kindly provided by Dr. Zhengli Shi, Wuhan Institute of Virology), recombinant anti-GAPDH antibody (Abcam, USA), and recombinant anti-actin antibody (Abcam, USA) at 4°C overnight, washed three times with 1 × TBST, and incubated with corresponding peroxidase-conjugated (Thermo Fisher Scientific) or fluorescently labeled (IRDye 680RD or 800CW: Li-Cor Biosciences) secondary antibody. The membranes were washed three times with 1 × TBST, and then scanned on an Odyssey Infrared Imaging System (Li-Cor Biosciences).