Clarity western ecl substrate reagent

Cell lysates were harvested from A549 cells, and proteins were resolved by SDS-PAGE and electro transferred to PVDF membranes where primary antibodies against E-cadherin (1:2000; #610181, BD Biosciences, San Jose, CA, USA), N-cadherin (1:1000; #610921, BD Biosciences, USA), and vimentin (1:500; sc-6260, Santa Cruz Biotechnology, Dallas, TX, USA) were used. Corresponding secondary horseradish peroxidase-conjugated antibodies (1:2000; Santa Cruz Biotechnology, Dallas, TX, USA) were used to detect primary antibodies on the membrane. Membranes were developed using BioRad Clarity Western ECL Substrate reagent in BioRad ChemiDoc imaging system (BioRad, Carlbad CA, USA). Protein levels were quantified by densitometry using Fiji open-source software (Image J version 1.52p; National Institutes of Health, Bethesda, MD, USA). Whole blots can be found in the Supplementary Materials (Figure S6).

Twenty-four hours after transduction with one of the recombinant viruses—rMVA-k1, rMVA-k2, rMVA-k5, rMVA-M001, rMVA-hlHA or rMVA-NP+M1 (multiplicity of infection (MOI) was 2)—BHK-21 cells were collected, washed three times with PBS, resuspended in RIPA lysis buffer containing 1% NP-40 and lysed using ultrasonication. Cell debris was removed by centrifugation (15,000× g, 15 min, 4). A PageRuler Plus protein marker (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the cell lysates were subjected to 12% SDS–PAGE (each sample contained 20 micrograms of protein). After electrophoresis under reducing conditions, the proteins were transferred to an Immun-Blot LF PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using wet electrophoretic transfer. The membranes were blocked in PBS containing 5% skim milk powder and 0.1% Tween 20 and then incubated overnight at +4 °C with antibodies against the 6x-His tag (HIS. H8-HRP, Invitrogen, Carlsbad, CA, USA) at a dilution of 1:20,000 or antibodies against beta-actin (AC-15-HRP, Abcam, Cambridge, UK) at a dilution of 1:50,000. Then, the membranes were washed 3 times in PBS with 0.1% Tween 20. Protein complexes were detected using Clarity Western ECL Substrate reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in accordance with the manufacturer’s recommendations and visualized using X-ray film (Fujifilm, Tokyo, Japan).

Total protein of MAC-T cells was extracted using the Total Protein Extraction Kit (Sango Biotech, Shanghai, China) for the detection of Nrf2 and Keap1 protein expression. The BCA protein assay kit was used to measure total protein concentrations, and 20 μg protein was separated by SDS-PAGE (Genscript, Nanjing, China) at 140 V for 45 min and electrophoretically transferred to polyvinylidene fluoride membranes (GE Healthcare, Wasukesha, WI, USA). Membranes were blocked with 5% skim milk buffer for 1 h, and then incubated overnight at 4 °C with primary antibody against Nrf2, Keap1 or GAPDH (Proteintech, Wuhan, China). Subsequently, membranes were washed 3 times with Tris-HCl Tween buffer (TBST) and incubated at room temperature for 1 h with secondary antibody (HRP-conjugated Affinipure Goat Anti-Mouse IgG, Proteintech, Wuhan, China). Then membranes were washed 3 times with TBST, and then were developed with the Clarity Western ECL Substrate Reagent (Bio-Rad, Hercules, CA, USA). Finally, the membranes were visualized using the ChemiDocTM MP System (Bio-Rad, Herculers, CA, USA), and the band densities was measured using Image Lab 6.0.1 software (Bio-rad, Herculers, CA, USA).