Concanavalin a cona agarose

For a glycoprotein analysis of hMRC1, exogenously expressed untagged hMRC1 was purified from transfected HEK293T cells as follows: Cells were washed once with PBS and lysed in 500 µL of TritonX-100-based lysis buffer. The samples were incubated in lysis buffer for 20 min at 4 °C on a rotator. Insoluble material was then removed via centrifugation at 15,000 rpm for 10 min. Ten percent of the clarified supernatant was used as an input control. The remaining lysate was incubated for 2 h at 4 °C on a rotator with Concanavalin A (ConA) agarose (Vector Laboratories, Burlingame, CA, USA; Cat# AL-1003). ConA-hMRC1 complexes were washed three times with TritonX-100 lysis buffer. Half the samples were left untreated, while the second half was treated with endoglycosidase H (EndoH). For an EndoH analysis, digestion was performed directly on hMRC1 bound to ConA agarose. All reagents and buffers were provided by the manufacturer (New Engaland Bio Labs, Ipswich, MA, USA; Cat# P0702). Samples were first washed once with denaturing buffer, then suspended in denaturing buffer, and heated at 95 °C for 10 min. Glyco buffer was then added along with an excess of enzyme (2500 units of Endo H), and samples were incubated at 37 °C for 1 h. Bound proteins were eluted from beads by boiling in an equal volume of sample buffer for 10 min at 95 °C and analyzed via immunoblotting.

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.