Sc 46692

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.

HeLa cells, cultured in DMEM (10% FBS 1% Pen/Strep), were plated in 6‐well plates. 24 h later, cells were counted and transduced with vector particles at a particle per cell ratio of 1,000. 24 h later, cells were harvested by extensive trypsin treatment and PBS washing steps to remove any membrane bound vector particles. Cells were counted and 3–5 × 10⁵ cells were used for subcellular fractionation, while 10⁵ cells were analysed by flow cytometry (CytoFlex S platform, Beckman Coulter) to determine transduction efficiency. Subcellular fractionation was performed as previously described (Rossi et al, 2019). Membrane, cytosolic and nuclear fractions were collected. Purity of fractions was confirmed by Western blot using anti‐Rab 5 (1:100, sc46692, Santa Cruz), anti‐Tubulin (1:5,000, T5198, Sigma Aldrich), anti‐Lamin B1 (1:5,000, 16048, Abcam) and anti‐Calreticulin (1:100, PA3‐900, Affinity BioReagents) antibodies. Fractions were subjected to DNA isolation (Qiagen, DNeasy Tissue kit) followed by qPCR analysis (FastStart essential DNA green master reagent, Roche) using CMV promoter‐specific primers on the LightCycler 96 real‐time PCR system (Roche). The specificity of target DNA amplification was confirmed by melting‐curve analysis. All samples were run in technical duplicates.