Plent ef1a fh cmv gfp p2a puro

The ORF of mouse Irgm1 (Myc-DDK-tagged) and Cul4b (Myc-DDK-tagged) were purchased from Origene and cloned into the lentiviral expression vector (pCDH-CMV-MCS-EF1-Puro, one kind gift of Dr. Jupeng Yuan, Shandong Cancer Hospital and Institute, China) or the His-Tag lentiviral expression vector (pLent-EF1a-FH-CMV-GFP-P2A-puro purchased from vigene).
For transfection, HEK293T cells were cultured to the concentration of 30%. Transfection was performed in 500 μl opti-DMEM with 20 μl siRNA or control with addition of 30 μl X-tremeGene (Roche, Mannheim, Germany) according to the manufacturer’s instruction. 2 μg constructions for ectopic expression of CUL4B, IRGM1 and HA-Ubiquitin were transfected by PEI transfection reagent (Merck) as indicated respectively. After 48 h incubation, MG132 was added at a final concentration of 10 μM. The cells were then harvested after 3 h incubation.

The BPV-13 E5 fragment (GenBank accession no. KM258443.2, Pang et al., 2014 (link)) was cloned into the AsisI and MluI sites of lentiviral plasmid pLent-EF1a-FH-CMV-GFP-P2A-Puro (Vigenebio, Shandong, China). The C-terminus of E5 was fused with a flag-6×his tag (Fig. S1). This recombinant plasmid pLent-EF1a-E5-FH-CMV-GFP-P2A-Puro and the control plasmid were co-transfected with PMD2G and PSPAX2 packaging plasmids into HEK293T cells, respectively. After 72 h, the supernatant was collected for purification by ultracentrifugation. The purified recombinant lentivirus and control lentivirus with a high-titer of 1.0 × 10⁸ TU/mL (transducing units/milliliter) were collected and stored at −70 °C.

The optimized E6 fragment was cloned into the Asis I and Mlu I sites of lentiviral plasmid pLent-EF1a-FH-CMV-GFP-P2A-Puro (Vigenebio, Shandong, China). The C-terminus of E6 was fused with a flag-6 ×his tag (Fig. S2). This recombinant plasmid pLent-EF1a-E6-FH-CMV-GFP-P2A-Puro and the control plasmid were co-transfected with PMD2G and PSPAX2 packaging plasmids into HEK293T cells, respectively. After 72 h, supernatant was collected for purification by ultracentrifugation. Purified recombinant lentivirus and control lentivirus with a high-titer of 1.0 × 10⁸ TU/ml (transducing units/ milliliter) were collected and stored at −70 °C.