Agarose a g beads

Cells were lysed in RIPA buffer and protein concentrations were determined by a BCA Protein Assay Kit (Santa Cruz, CA, USA). Immunoblotting were performed as described previously [22 (link)]. For immunoprecipitation, extracts were pre-cleared with 30 μL agarose A/G beads (Beyotime Institute of Biotechnology) by rotating for 1 h at 4°C. Beads were removed, and then another 30 μL agarose A/G beads and 1.5 μg antibodies were added to the lysates rotating overnight at 4°C. The beads were then washed twice in basic lysis buffer, and boiled for 10 min in loading buffer before resolving by SDS-PAGE. The blots were detected by an imaging system (Bio-Rad, USA).

Vero cells transfected with pcDNA3.1(+)-PABPC1 plasmid with FLAG-tag were infected with PEDV at an MOI of 0.1 and harvested at 6 hpi. The total proteins were extracted as described above. The supernatants were incubated with anti-FLAG mouse monoclonal antibody for 12 h at 4 °C, then Agarose A+G beads (Beyotime, Shanghai, China) were added. After 6 h incubation, the beads were collected by centrifugation at 2500× g for 5 min and washed eight times with cold protein extraction buffer. The beads were boiled in 5× SDS loading buffer to elute bound proteins for mass spectrometry analysis.
To assay the interaction between PABPC1 and PEDV N protein, 293T cells were transfected with pcDNA3.1(+)-PABPC1 or empty plasmid with FLAG-tag and pcDNA3.1(+)-PEDV-N or empty plasmid with HA-tag, respectively. After 24 h, the cells were lysed and incubated with anti-FLAG monoclonal antibody, and the eluted samples were analyzed with anti-FLAG, anti-HA, or anti-PEDV antibodies.

Cells were lysed in RIPA buffer, and protein concentrations were determined using a BCA Protein Assay Kit (Santa Cruz, CA, USA). Proteins were immunoblotted using a standard protocol. For immunoprecipitation, extracts were precleared with 30 μL of agarose A/G beads (Beyotime Institute of Biotechnology) by rotary mixing for 1 h at 4°C. The beads were removed and another 30 μL of agarose A/G beads and 1.5 μg of the antibodies were added to the lysates. The mixture was then incubated on a rotary shaker overnight at 4°C. The beads were then washed twice in basic lysis buffer and then boiled for 10 min in loading buffer before loading on an appropriate SDS-PAGE. The blots were detected using an imaging system (Bio-Rad, USA).