Ecl reagent

We verified the protein expression of MMP14, PCDH7, and LAMP3 using immunohistochemical staining images from the HPA database (https://www.proteinatlas.org/). Moreover, we also verified the differential expression of CCL20, BTG2, IL7R, and TLR2 between normal bronchial epithelial cell line (16-HBS) and lung adenocarcinoma cell line (A549) by utilizing Western blotting. 16-HBS and A549 cell lines were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Protein concentrations were evaluated through the BCA Assay kit (CWBIO). Western blotting was performed as seen previously (Wang et al., 2020 (link)). Briefly, 15 ug proteins were loaded onto 8–15% polyacrylamide gels. After transfer, the NC membranes (Boster) were blocked with 5% skimmed milk at room temperature for 2 h. Next, membranes were incubated with primary antibodies [CCL20 (Affinity, 1:300), BTG2 (proteintech, 1:300), IL7R (Affinity, 1:300), TLR2 (proteintech, 1:1000)], and GAPDH (Elabscience, 1:3000) for 2 h at room temperature and then at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (CWBIO, 1:1000) at room temperature for 2 h. Finally, the bands were exposed with ECL reagents (CWBIO). ImageJ software was applied to perform the gray value analysis.

Mycobacteria were lysed by beads beating in 25 mM Tris-HCl, 100 mM NaCl and 1 mM EDTA with protease inhibitor (Roche). Lysates for Fig. S4 were quantified by Bradford assay kit (Bio-Rad), loaded in SDS-PAGE gel and immunoblotted with HRP-conjugated anti-GFP antibody (Clone LGB-1, CWbio) or HRP-conjugated anti-rpoB antibody (clone 8RB13, Santa CruZ Biotechnology). Blots were visualized using ECL reagents (CWbio).

MARC-145 cells were infected with a MOI of 0.1, and lysed in SDS loading buffer at 8, 16, 24, 48, 72, and 96 h post infection (hpi). The proteins in cell lysis solution were separated by SDS-PAGE, and transferred onto a PVDF membrane. After blocking with 5% skimmed milk overnight at 4°C, the membranes were, respectively, incubated with anti-NSP7α and anti-NSP7β polyclonal antibodies, followed by incubation with goat-anti-rabbit IgG (CWBIO, China) conjugated horseradish peroxidase as secondary antibody. Immunodetection was performed using enhanced chemiluminescence (ECL) reagents (CWBIO, China) according to the supplier’s instruction.

Total cell lysates were prepared by lysing cells in NETN buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40) with protease inhibitors (Merck) on ice for 30 min. The lysates were cleared by centrifugation at 16000 rpm for 10 min. The samples for western blotting were prepared by mixing the supernatant with equal volumes of 2× sample loading buffer (100 mM Tris–HCl pH 6.8, 100 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol). Equal amounts of total protein were separated by SDS–polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (PALL) and immunoblotted with antibodies. The membrane was further washed with TBST and developed using ECL reagents (CWBIO).

The cells were starved in an FBS-free medium for 24 h and then cultured in the absence or presence of arecoline for 48 h. The proteins were harvested using lysis buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime Bio, Wuhan, China). Protein concentrations were examined and proteins were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated with the antibodies to TPM1 (RabMAb, 1:2000, Catalog Number ab133292; Abcam, Cambridge, MA, USA), Smad2/3 (1:750, Catalog Number WL0152; Wanleibio, Shenyang, China), and p-Smad2/3 (1:500, Catalog Number WL02305; Wanleibio). Alpha-tubulin antibody (1:5000, Catalog NumberRM2007; Rayantibody, Beijing, China) was used as an internal control. Chemiluminescent signals were visualised using ECL reagents (Cwbiotech, Beijing, China).

To investigate the antigen binding of the generated MAbs, virus concentrated by ultracentrifugation was resuspended in reducing or non-reducing lane marker sample buffer (Thermo scientific, USA) and boiled for 6 min before SDS-PAGE separation. The separated proteins were transferred onto a PVDF (Polyvinylidene Fluoride) membrane, followed by incubation in blocking buffer (5% skim milk in PBS with 0.05% Tween-20) overnight at 4 °C. After washing, the protein was probed with the MAb and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG at a dilution of 1:5000. The signal was developed with chemiluminescence substrate (ECL reagent, Cwbiotech, Beijing, China). To further analyze the MAb binding domain, full length E protein of TMUV, domain I/II and domain III of E protein were individually expressed in E. coli using the pET32α vector (see Additional file 1). Purified and renatured recombinant protein was separated by SDS-PAGE under non-reducing condition and analyzed using the generated MAbs by Western blot as described above.