Plasmid-Encoded Protein Expression Protocols

Expression plasmids encoding CD63 and syntenin-1 were described previously50 (link)80 (link)81 (link). CD63/T7 was generated using a standard PCR protocol generating a CD63 protein which resembles the C-terminus of Tspan7 (primers are available upon request). CD63 was amplified from clone IRAUp969A121D6 (imaGenes, Berlin, Germany) by standard PCR. The CD63 sequence was then cloned into the XhoI-KpnI site of the pEGFP-C1 (Clontech, Heidelberg, Germany) vector. The ALIX-HA containing plasmid (pCAGGS-HA-AIP1) was a kind gift from Prof. Takemasa Sakaguchi (Hiroshima, Japan)82 (link). The ALIX V-domain containing plasmid (pGST2-ALIX^364–716) was a kind gift from Prof James Hurley (NIH, USA). The HPV16 L1-specific antibodies mouse mAbs L1-7, H56.E, and 16L1-312F and rabbit pAb K75 (detecting HPV16, 18, and 31) have been described previously22 (link)24 (link)29 (link)47 (link)83 (link). The CD63 antibody 6H1 was described previously84 (link), the CD63 mAb sc-5275, rat CD63 mAb AD1, syntenin-1 mAb sc-100336, Rab5 mAb sc-46692, mouse Golgin-97 mAb A-21270, ALIX mAb sc-53540, rat HA Ab 3F10 and the mouse mAb anti β-Actin (AC-15) were purchased from Santa Cruz (Dellas, TX, USA), Molecular Probes (Eugene, OR, USA), Roche (Basel, Switzerland), and Sigma-Aldrich (St. Louis, MO, USA), respectively. The LBPA mAb was kindly provided by Jean Gruenberg, Geneva, Switzerland.