Clarity ecl substrate

The whole-cell lysates or the nuclear lysates were run on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and were transferred to polyvinylidene difluoride membrane using wet transfer. After the transfer, the membrane was incubated with 5% (w/v) bovine serum albumin (BSA) in TBST [20 mM Tris buffer pH 7.5, 150 mM NaCl, and 0.1% (v/v) Tween 20] for blocking. Then, it was incubated with various antibodies purchased from different companies. The antibodies against IκB, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), p65, c-Rel, and Lamin B1 were purchased from Santa Cruz Biotechnologies, USA. The antibodies against p-p38, p38, phosphorylated c-Jun N-terminal kinase (p-JNK), JNK, IκB kinase α/β (IKKα/β), Cullin-1, Cand-1, β-TrCP, Skp-1 and Rbx-1 were purchased from Cell Signaling Technologies, USA. The antibodies against His-tag, HA-tag, and proliferating cell nuclear antigen (PCNA) were purchased from Biolegend, USA. The antibody against Nedd8-Cullin was purchased from Abcam, UK. The antibody against LAMP-1 was purchased from Thermo Fisher Scientific, USA.
The horseradish-peroxidase-tagged secondary antibodies and antibody against glutathione-S-transferase (GST) were purchased from Sigma-Aldrich, USA. The immunoblots were developed using Clarity^TM ECL Substrate (Bio-Rad, USA) and detected using LAS 4000 (GE Healthcare Technologies, USA).

Total protein extracts were obtained by lysing the cells with hot Laemmli buffer (60 mM Tris-HCl pH 6.8, 100 mM DTT, 5% glycerol, 1.7% SDS) and passed through syringes (26G) [3 (link)]. Cytosolic and nuclear protein extracts were isolated from mesothelioma cells, and their purity was assessed as previously described [20 (link)]. A total of 5 μg of protein extract was separated on denaturing 15% SDS-PAGE gels, and proteins were transferred onto PVDF membranes (0.45 μm, Perkin Elmer, Waltham, MA, USA). Membranes were probed with the following primary antibodies: RBM8A (Sigma-Aldrich, St. Louis, USA, cat. #HPA018403, RRID:AB_1858908), ADAR1 (Sigma-Aldrich, cat. no. HPA003890, RRID:AB_1078103), ADAR2 (Santa Cruz Biotechnology, Dallas, TX, USA, cat. no. sc-73409, RRID:AB_2289194), MSI (Cell Signaling Technology, Danvers, USA, cat. no. 85652, RRID:AB_2800060), and mouse anti-β-actin (C4, MP Biomedicals MP691002 RRID:AB_2335127). Membranes were then incubated with one of the following secondary antibodies: rabbit anti-mouse IgG-HRP (no. A9004) or goat anti-rabbit IgG-HRP (no. A0545), obtained from Sigma Aldrich. The signals were detected by enhanced chemiluminescence (Clarity TM ECL Substrate, BioRad, Hercules, CA, USA) using Fusion Digital Imager (Vilber Lourmat, Marne-la-Vallée, France). Quantification was carried out using ImageJ software, version 1.52a.

To detect the target proteins through western blotting, the proteins separated by SDS PAGE were transferred to a PVDF membrane (Bio-Rad) using a semidry transfer device (Bio-Rad). Membranes were incubated with TBST solution (Tris-buffered saline with 0.1% Tween® 20 detergent) containing primary antibodies. Anti-GFP (Thermo) and anti-TAP (Thermo) were purchased; anti-Act1 was derived from the Taiwan yeast resource center. anti-uL8 and anti-eS24 were generated in this lab. Protein signals were detected by Clarity^TM ECL Substrate (Bio-Rad). Images were acquired with MultiGel-21 (TopBio, Taiwan).

Whole cells lysates for assessing relative protein level or gel mobility were prepared using a TCA-precipitation-based method as previously described with slight modification^{62 (link)}. Precipitated protein pellets were rinsed in cold acetone and resuspended in a modified loading buffer (40 mM Tris-HCl pH 6.8, 8 M urea, 5% SDS, 0.1 M EDTA), which did not contain β-mercaptoethanol or bromophenol blue. Samples were diluted 1:20 in 1% SDS to measure protein concentration by DC protein assay kit using BSA as a standard. Samples were diluted to the same protein concentration and mixed with 1/5 volume of the modified loading buffer supplemented with 0.6% (v/v) β-mercaptoethanol and ~0.3 mg/mL bromophenol blue. Total protein (~30 µg) in samples was resolved on 10% SDS-gels and blots probed using anti-Flag (F3165; Sigma; 1:3000) or anti-HA (3F10; Roche; 1:3000) primary antibody and the corresponding HRP-conjugated secondary antibody. Blots were developed using Clarity^TM ECL substrate (Bio-Rad) and chemiluminescent signals captured by ChemiDoc Imager (Bio-Rad).

Cells were washed in PBS and then lysed in buffer containing 1% Triton X-100, 0.1% NP-40, 150 mM NaCl, and 10 mM Tris-HCl (pH 7.4) supplemented with a protease inhibitor cocktail (Cell Signaling). Protein concentration was determined using a bicinchoninic acid (BCA) assay following the manufacturer’s protocol (Thermo). Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Proteins were detected using appropriate primary antibodies (1 μg/ml concentration) and horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using chemiluminescence according to manufacturer protocol (Bio-Rad Clarity ECL substrate).

Total RNA was extracted from purified virions using the TRIzol reagent. Virion RNA (5 ng) was denatured and separated on 1.5 or 1.7% denaturing agarose-formaldehyde gels and transferred onto a nylon membrane with a molecular weight marker (BioDynamics Laboratory). Biotin-labeled strand-specific RNA probes for HA, NA, and NS vRNAs and 18S and 28S rRNAs were synthesized using a biotin-11-UTP (Roche) and a T7 RNA Expression Kit (Promega) according to the manufacturer's instructions. Northern blot analyses were performed using an ABC Kit (Vector) and a DIG block and wash buffer set (Roche) according to the manufacturer's instructions. The signals were detected with Clarity ECL substrate (Bio-Rad). The uncropped scans of the blots are shown in Supplementary Fig. 1.