Peroxidase affinipure donkey anti mouse igg h l

The protein samples were electrophoresed on a 4–12 % Bis-Tris gel (Life Technologies), followed by standard western blotting protocols. Primary antibodies used were as follows: anti-TERT antibody (Abcam, ab32020, 1:1000), anti-GAPDH antibody (Santa Cruz, sc-137179, 1:1000), anti-FLAG horseradish peroxidase-conjugated antibody (Sigma-Aldrich, A8592, 1:1000). Secondary antibodies used were as follows: peroxidase-AffiniPure donkey anti-rabbit IgG (H + L) (Jackson, 711-035-152, 1:5000), peroxidase-AffiniPure donkey anti-mouse IgG (H + L) (Jackson, 715-035-150, 1:5000). SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific) was used to generate signals on western blots. The signals were detected with a FluorChem HD2 imaging system (Alpha Innotech) and quantified with ImageQuant TL v2005 software. To detect the SNAP-tag, 10 μM SNAP Surface® 594 (New England Biolabs, S9112S) was added to the input samples at the beginning of the IP. The fluorescence signals were detected with a Typhoon Trio PhosphorImager (GE Healthcare) and quantified using ImageQuant TL v2005 software.

Vero A/T cells were prepared as in Section 2.3 and infected with SARS-CoV-2 (England-2) at an MOI of 5 for 30 min on a platform rocker at 37 °C, 5% CO₂. Cells were subsequently treated with 6.4 mg of product, as in Section 2.3. At 24 and 48 hpi, supernatant was removed and cells were fixed via the addition of 0.5 mL/well 4% (v/v) formaldehyde for 30 min. Cells were washed twice with PBS and 250 µL/well of NP-40 was added for 15 min to permeabilise cells. Cells were washed once with PBS-Tween (PBST) and blocked in 3% milk for 1 h in the dark. Block was removed and cells were stained with 250 µL/well of anti-nucleocapsid (N) primary antibody (SARS-CoV-2 Nucleocapsid Protein (1C7) Monoclonal Antibody; bsm-441411M; Generon, Slough, UK) and diluted in 1% milk in PBST for 1 h in the dark. Cells were washed once with PBST and 250 µL/well of secondary antibody (Peroxidase AffiniPure Donkey Anti-Mouse IgG (H + L); Jackson ImmunoResearch Europe Ltd., Ely, UK) was added in 1% milk in PBST for 1 h in the dark. Cells were washed once with PBST and levels of N were determined by the OD at 492 nm following the addition of OPD substrate as per manufacturers’ instructions (OPD Substrate Tablets; ThermoFisher Scientific, Waltham, MA, USA).

Fifty microgram protein in Ripa buffer and sample buffer from 2× stock (62.5 mM Tris–HCl pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 10% β‐mercaptoethanol) was denatured for 5 min at 95°C. Samples were loaded on 10% or 12% SDS–PAGE gels and migrated at 80 V for 15 min, then 120 V and transferred on nitrocellulose (88018 Thermo Fisher) at 15 V for 45 min. Proteins were stained by Ponceau staining. Membranes were washed in TRIS‐buffer saline containing 0.1% Tween (TBST) (100 mM Tris base, 3 M NaCl, 2% Tween 20) and blocked for 30 min in 5% non‐fat dried milk in TBST, except membranes probed with phospho‐specific primary antibodies which were blocked in 5% bovine serum albumin (A9647 Sigma‐Aldrich) in TBST. Membranes were incubated overnight at 4°C with monoclonal mouse anti‐Memo1 antibody (Haenzi et al., 2014 (link)) kindly provided by Prof. Olivier Bonny, University of Lausanne, washed with TBST, and incubated with 1:10,000 Peroxidase AffiniPure Donkey Anti‐Mouse IgG (H+L) (715035‐150 Jackson ImmunoResearch) for 1 h at room temperature and washed with TBST. Signals were detected using Pierce ECL Western Blotting Substrate (32106 Thermo Fisher) and developed on ChemiDoc XRS+ System (Bio‐Rad Laboratories, Inc.). Bands were quantified using ImageJ.