Pierce ecl immunoblotting substrate

Proteins were separated on 4–15% Mini-Protean TGX precast gels (BioRad). Molecular weight markers (PageRuler^TM Prestained Protein Ladder) were from Thermo Scientific. Gels were stained with Bio-Safe Coomassie G-250 (BioRad, Solna, Sweden ) or, when used for immunoblots, transferred onto 2 µm PVDF membranes (BioRad). Membranes were blocked for 1 h in 5% non-fat powdered milk in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) followed by incubation for 1 h with primary antibody in 1% non-fat powdered milk in PBS. Membranes were then washed three times with PBST (PBS plus 0.1% Tween-20) followed by 1 h incubation with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (BioRad) in 3% non-fat powdered milk in PBS. The membrane was then washed twice with PBST followed by one wash in PBS before being developed using a Pierce ECL immunoblotting substrate (Thermo Scientific, Stockholm, Sweden) and imaged using a ChemiDoc MP system (BioRad).

Cells were seeded in 6-well plates and infected with SARS-CoV-2 after incubation for 24 h. At various time points as indicated, cells were collected, lysed and protein concentration was determined by BCA protein assay kit (Beyotime, P0010). After mixed with loading buffer and boiled for denaturing, equal amounts (30 μg) of proteins were electrophoresed, transferred into a membrane and then incubated overnight at 4°C with specific primary antibodies. After further incubation with HRP-conjugated secondary antibodies, the specific bands were visualized with the Pierce ECL Immunoblotting Substrate (Thermo Fisher Scientific, 32106) using the Amersham Imager 600 system. In siRNA gene silencing experiments, cells were collected without SARS-CoV-2 infection and subjected to the immunoblotting procedure described above.

A549 2x10⁵ cells were cultured in 2% DCC-RPMI medium for 24 h. Cells were pre-treated with BR2-2xPPD or BR2-2xΔPPD 2.5 µM for 4 h, then induced with EGF 20 ng/ml for 5, 10, and 30 min to activate MAPK signaling. Cells were washed with cold PBS and lysed using lysis buffer (5 mM NaF, 2 mM Na₃VO₄, 1xproteinase inhibitor in RIPA buffer). Cell lysates were mixed with 4x Laemli loading buffer, loaded on 8% SDS-PAGE gels and transferred onto PVDF membranes.Membranes were probed with primary antibodies recognizing phospho-p44/42 MAPK (1:1000 v/v, Cell Signaling Technology, USA) and total MAPK antibody (1:1000 v/v, Cell Signaling Technology, USA) overnight at 4°C. Next, blots were incubated with an anti-rabbit secondary antibody (1:5000 v/v in 5%BSA-TBST) for 1 h at room temperature and visualized by chemiluminescence using Pierce® ECL Immunoblotting Substrate (Thermo Scientific, USA). Images were analyzed by ImageJ software. All Western Blots shown were representative of at least three independent experiments.