Xcell 2 blot module

NuPage Novex 4–12% Bis-Tris gels (Invitrogen) were used for all SDS-PAGE experiments. Following SDS-PAGE, proteins were transferred to nitrocellulose membrane using the XCell II™ Blot Module (Invitrogen) following the manufacturer's protocol. Protein bands were visualised with a quantitative infrared imaging system (LI-COR Odyssey).

Proteins were separated by SDS-PAGE electrophoresis on NuPAGE 10% Bis-Tris Gels (Invitrogen) using MES-SDS running buffer (50 mM MES, 50 mM Tris-base, 3.5 mM SDS, 1 mM EDTA, pH 7.3) under reducing conditions. Gels were visualized by Coomassie blue staining. For Western blot analysis, proteins were transferred to PVDF membranes (Amersham Hybond™-P, GE Healthcare) by semi-wet blotting (XCell II™ Blot Module, Invitrogen, Life Technologies) following the manufacturer’s instructions. Blots were blocked with 2% ECL Prime blocking agent (GE Healthcare) in PBS-T [PBS buffer supplemented with 0.1% (v/v) Tween-20]. For anti-SARS-CoV-2 antibody detection, the blots were incubated with 1:20,000 HRP-conjugated rabbit anti-human IgG (Sigma-Aldrich, #A8792). For SARS-CoV-2 antigen detection, the blots were incubated with 1:2,000 Anti-His mouse monoclonal primary antibody (Qiagen, #34660) and then incubated with 1:10,000 peroxidase labeled anti-mouse IgG secondary antibody (GE Healthcare). Blots were developed with ECL Prime Western blotting Detection Reagent (GE Healthcare) and visualized using a Fujifilm LAS-3000 imager.

For Western blot analysis, lysosomal extracts (1–5 μg total protein) were separated by SDS-PAGE (NuPAGE 4–12% Bis-Tris, Invitrogen) and stained with Coomassie SimplyBlue Safe Stain (Invitrogen) or Ponceau S or transferred to PVDF (0.45 μm) membranes for Western blot analysis. Transfer conditions using the XCell II Blot Module (Invitrogen, EI9051) with a constant 25 V, 125 mA for 90 min. Posttransfer PVDF membranes were fixed for 30 min with 4% paraformaldehyde in PBS at room temperature. Blots were blocked in Tris-buffered saline containing 0.1% Tween 20 (Sigma) and 5% nonfat dry milk (170–6404, Bio-Rad) for 1 h at room temperature. The blocked membrane was incubated in blocking buffer containing the following v/v of primary antibodies: Syn-1 (1:1000), EP1646Y (1:1000), ab138501 (1:2000), LB509 (1:5000), ab6176 (1:1000), ab53726 (1:1000), Syn211 (1:2000), ab52168 (1:1000), ab21976 (1:5000), and ab59264 (1:5000) overnight at 4 °C, followed by four 10-min washes. Membranes were incubated in blocking buffer containing HRP-conjugated secondary antibody (1:10,000) for 1 h at room temperature. Blots were developed using ECL chemiluminescent substrate (Thermo Fisher). Film (876 1520, Kodak) was exposed for varying times between 10 s to 5 min. All blots were performed at least two times for each biological replicate.

Protein was extracted on ice from embryos at E10.5 using RIPA buffer. Protein concentrations were determined using the Bradford assay. Western blot was performed using conventional methods, with 50-μg protein run per sample on NuPAGE 4–12% Bis-Tris gel (Life technologies), followed by transfer to PVDF membranes (XCell II Blot Module, Invitrogen). The primary antibodies were rabbit anti-caspase-8 (1:500, Proteintech, Chicago, USA) and anti-β-TUBULIN (1:10,000, Proteintech, Chicago, USA). After incubation with secondary antibody (1:10,000, DAKO), blots were developed using ECL Prime (GE Healthcare Life Sciences) or ECL Western Blotting Substrate (Promega). ImageJ was used to detect densitometry. The results were normalized using β-TUBULIN loading control. Independent experiments were carried out three to four times per sample.

Cells were re-suspended in NuPAGE sample buffer supplemented with 50 mM of dithiothreitol (DTT) and loaded on 4–12% NuPAGE Bis-Tris Protein Gels and electrophoresed at 180 V for 1 h. Gels were transferred to Nitrocellulose membranes using wet transfer system XCell II Blot Module (Invitrogen) at a constant amperage of 350 mA for 2 h. Membranes were blocked with 5% skimmed milk in Tris Buffered saline-Tween for 1 h before incubation with primary antibody anti-myc tag monoclonal clone 4A6 (Millipore) diluted to a final concentration of 200 ng mL⁻¹ in 3% skimmed milk in TBST for 1 h at room temperature. After washing membranes proceeded to incubation with secondary antibody anti-mouse IgG (H + L) conjugated to HRP (Molecular Probes) at 300 ng mL⁻¹ in 3% skimmed milk in TBST for 1 h at room temperature. Membranes were washed, incubated with Clarity Max substrate (BioRad) and developed in ChemiDoc MP (BioRad).

A549 cells were infected with rRSVflag(2)L at an MOI = 3. At 20 hpi, cells were lysed in RIPA lysis buffer and genomic DNA was digested with TURBO DNaseI (Invitrogen, AM2238). 5% of the sample was run on a tris-glycine gel and was transferred to a nitrocellulose membrane using XCell II blot module (Invitrogen) for 2 hrs at 30V in a transfer buffer containing 10% methanol. Blot was analyzed with primary antibody ANTI-FLAG(R) M2 (Sigma-Aldrich, F3165) at a dilution of 1:500 and secondary antibody IRDye 800CW anti-Mouse IgG (Licor, 26–32212) at a dilution of 1:20,000.