Supersignal west femto maximum sensitivity chemiluminescent substrate kit

For Western blotting, transfected cells were lysed in buffer containing 100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 20% β-mercaptoethanol and boiled for 5 min. Then, proteins in cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Subsequently, membranes were blocked for 1 h in PBS containing 5% dried skim milk and 0.1% Tween-20 and incubated with an anti-HA polyclonal antibody (Sigma, Stockholm, Sweden) or an anti-actin monoclonal antibody (Sigma) at 4 °C overnight. Horseradish peroxidase (HRP) secondary antibodies (Merck life science, Darmstadt, Germany) specific for either mouse or rabbit immunoglobulins (Ig) were used to detect bound primary antibodies. Proteins in the membranes were detected with a SuperSignal West Femto maximum-sensitivity chemiluminescent substrate kit (Thermo Scientific) following the manufacturer’s instructions.

Transfected cells were lysed in RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and proteins were separated by denaturing electrophoresis using 10% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. Membranes were blocked for 1 h with 5% dried skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (T-PBS) and incubated overnight at 4 °C with primary rabbit polyclonal antibodies (pAb) against NS1 [53 (link)] or a mix of mouse monoclonal antibodies (mAb) against PA (1F6, NR-19225; 5C5, NR-19226; and 8E10, NR-19224) obtained from BEI Resources. A mouse mAb against actin (A1978; Sigma) was used as an internal loading control. Horseradish peroxidase (HRP) secondary antibodies (GE Healthcare) against either mouse or rabbit immunoglobulins (Ig) were used to detect bound primary antibodies. Protein expression was detected with a SuperSignal West Femto maximum-sensitivity chemiluminescent substrate kit (Thermo Scientific) in accordance with the manufacturer’s instructions. Protein bands were normalized to the level of beta-actin expression, and then the level of expression of the WT protein was considered 100%. In the case of Figure 2C, for the anti-PA analysis, PA_WT+/NS1_MT− was considered 100%.