构建N-端表达VSVG的蛋白： To generate fusion constructs, cDNA fragments encoding the full-length mouse GPR56 protein were used as a template for PCR. For VSVG/His-GPR56, the primers were forward primer 5′-GGCTCCGGAGAGCCCCCGAGAAGACTTCCGCTTCTG-3′ and reverse primer 5′-CGCCGCGGCCGCTCAATGATGATGATGATGGATGCGGCTGGAGGAGGTGCTG-3′ (the reverse primer codes for a 6-His tag at the C-terminus of the protein).
利用Biotin捕捉VSVG：
Biotinylation of cell surface proteins was performed as described. Briefly, HEK293T cells were transfected with VSVG/His-tagged wild-type or mutant GPR56 constructs. Twenty-four hours after transfection, cells were washed with ice-cold PBS and incubated with 0.5 mg/ml of Sulfo-NHS-Biotin (Pierce) in PBS for 1 h at 4°C. The cells were subsequently washed extensively in PBS/glycine to remove any unbound biotin prior to lysis with RIPA buffer. Equal amounts of cell lysates were incubated with streptavidin–agarose beads (Sigma, St Louis, MO) for 2 h at 4°C. The beads were then washed three times in RIPA buffer, and the biotinylated proteins were eluted with 8 M urea (in 50 m M Tris, pH 8.0). Samples were further denatured in SDS sample buffer, separated by SDS–PAGE and transferred to a nitrocellulose membrane. The membranes were then probed with various antibodies, using standard western blot protocol.
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