β actin mab

Cells were lysed in RIPA buffer (50 mM Tris [pH 8.0], 5 mM EDTA, 150 mM NaCl, 10% glycerol, 0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 1% Triton X-100, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 25 mM NaF, 10 µg of pepstatin A/ml, 10 µg of aprotinin/ml, 25 µg of leupeptin/ml, 25 µg of trypsin inhibitor/ml). 20 µg of protein, determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific), were boiled for 5 min in presence of 2-mercaptoethanol, then separated by SDS-PAGE and immobilized on nitrocellulose membranes. Membranes were blocked for 15 min with 5% non-fat skim milk in Tris-buffered saline (20 mM Tris, 137 mM NaCl [pH 7.6]) containing 0.1% Tween-20 (TBS-T) and then incubated for 4 h with a pan-T antigen monoclonal antibody (mAb) (clone F4) (Pallas et al., 1986 (link)), rabbit polyclonal antibodies against VP2 and VP3(graciously provided by Robert Garcea, University of Colorado at Boulder), or β-actin mAb (Cell Signaling Technology) in TBS-T containing 1% milk. Blots were washed three times for 15 min each with TBS-T, followed by a 4 h incubation with HRP-conjugated secondary antibodies (Jackson ImmunoResearch) in TBS-T containing 1% milk. Blots were washed three times and developed with the ECL enhanced chemiluminescence system (ThermoFisher Scientific).