Anti c myc tag affinity gel

Cells transfected with recombinant plasmids were lysed in the lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, and 5% glycerol) containing 1 mM PMSF and protease inhibitor cocktail (TargetMol), and applied to centrifugation at 15,000 × g for 20 min at 4 °C. Cell lysates were subjected to precipitation using 20 μl ANTI-FLAG^® M2 Affinity Gel (A2220, Sigma), Anti-c-Myc Tag Affinity Gel (9E10, BioLegend), or Anti-HA Magnetic Beads (88836, Pierce) overnight at 4 °C. The sample was washed five times with the lysis buffer to remove unbound proteins and suspended in 1× SDS loading buffer. The suspended sample was boiled for 5 min at 100 °C before analysis by SDS-PAGE and western blotting with the indicated antibodies.

HEK293T, CNE2 and HNE1 cells stably expressing gB-SFB were amplified and subjected to TAP according to our previous methods[31 (link)]. MS analyses were performed by Beijing Proteome Research Center and Wininnovate Bio. (Shenzhen, China).
For immunoprecipitation, cells were lysed in NETN buffer (20 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing 50 mM β-glycerophosphate, 1 μg/mL pepstatin A and 10 μM leupeptin, and the bait proteins were precipitated by S-protein beads (Navagen), Streptavidin beads (Amersham) or anti-c-Myc Tag affinity gel (Biolegend) as indicated.
For the GST pull-down assay, GST or GST-FBXO2 recombinant protein immobilized onto glutathione Sepharose 4B beads (GE Healthcare) was incubated with gB protein purified from stably infected HEK293T cells in NETN buffer for 4 h at 4°C. For Con A pull-down, cell lysates were incubated with Concanavalin A (Con A) agarose (Vector Labs) for 4 h at 4°C. The bound proteins were eluted with 1X SDS loading buffer.