A0545 1ml

The level of protein in brain tissue homogenate was estimated and was separated by SDS‐PAGE. Then proteins were transferred to polyvinylidene membranes and incubated with primary antibodies for TTP (ab83579; Abcam, Table 1) and PP2A (ab16448; Abcam, Table 1) for 12 hr. The membranes were incubated with HRP‐IgG (goat anti‐rabbit, A0545‐1ML; Sigma‐Aldrich) for 1 hr. TTP and PP2A were assessed by enhanced chemiluminescence (ECL; Zhuang, Ye, & Huang, 2017).

Viral protein accumulation was analyzed by Western blot of Vero E6 cell lysates, previously transfected with 2.5 μg of circRNAs and infected with SARS-CoV-2 at an MOI of 0.1 pfu/cell at 33°C. Total protein lysates obtained at 24 h post-infection were heat-denatured in SDS-loading buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 2.5% 2-mercaptoethanol, and 0.05% bromophenol blue) at 95°C for 10 min. Following separation by SDS-polyacrylamide gel electrophoresis (PAGE; 10%), proteins were blotted onto a nitrocellulose membrane (BioRad). Viral proteins were immunostained overnight with rabbit anti-SARS nucleocapsid protein (N, 200-401-A50; Rockland Immunochemicals, 1:500) or mouse anti-GAPDH antibody (monoclonal antibody G8795, GAPDH-71.1; Sigma-Aldrich, 1:5000) and appropriate secondary antibodies (HRP-conjugated anti-rabbit (A0545-1ML) or anti-mouse (A9044-2ML), Sigma-Aldrich, each 1:10,000). The blots were developed using the Lumi-Light Western-Blotting Substrate (Roche). Western blot signals were estimated by densitometry, using GelAnalyzer 19.1 software.
For subcellular fractionation, 1.25% of cytoplasmic or nuclear fractions were analyzed by SDS-PAGE (10%) and Western blotting through detection of hnRNP A1 (monoclonal antibody, sc-32301, 4B10; Santa Cruz Biotechnology, 1:2000) and GAPDH (monoclonal antibody G8795, GAPDH-71.1; Sigma-Aldrich,1:5000).

Cell pellets corresponding to OD₆₀₀ 5–10 were protein extracted using the alkali extraction protocol described in [18 (link)], and samples corresponding to OD₆₀₀ 0.5–1 were analyzed by SDS-PAGE and western blotting (WB). Monoclonal mouse anti-His primary antibody (Sigma H1029-2ML, 1:5000 dilution) and anti-mouse-HRP secondary antibody (Santa Cruz Biotechnology sc-2060, 1:10,000 to 1:20,000 dilution), or polyclonal rabbit anti-NifU ([13 (link)], 1:5000 dilution), polyclonal rabbit anti-NifS ([13 (link)], 1:200 dilution), polyclonal rabbit anti-NifM (1:4000 dilution), and anti-rabbit-HRP secondary antibody (Sigma A0545-1ML, 1:10,000 dilution) were used.
After the immunodetection of proteins, polyvinylidene fluoride (PVDF) membranes were stained with Coomassie to determine the total protein loaded and the blotting efficiency, as described in [19 (link)].
Representative expression results are shown throughout the manuscript. For every figure, at least two transformant colonies were analyzed for each strain and the results were obtained from at least two biologically independent experiments.