Extended Data Fig. 9: The GFP-SAMHD1 fusion protein, expressed from the SAMHD1 locus, can be degraded by the lentiviral Vpx protein. | Nature Methods

Extended Data Fig. 9: The GFP-SAMHD1 fusion protein, expressed from the SAMHD1 locus, can be degraded by the lentiviral Vpx protein.

From: Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses

Extended Data Fig. 9

Monoclonal SAMHD1 KO cells were generated in human glioblastoma cell line LN18. Next, SAMHD1 KO LN18 cells were stably transfected with linearized plasmids encoding for either GFP-SAMHD1 or SAMHD1-GFP. GFP-positive cells were sorted by flow cytometry 4 weeks after transfection. To deliver Vpx into cells, WT LN18 and SAMHD1 KO cells expressing either GFP-SAMHD1 or SAMHD1-GFP were challenged with VSV- G pseudotyped virus like particles (VLPs) with incorporated lentiviral Vpx (Vpx-VLPs). Empty VLPs (Ø) or VLPs with incorporated Vpr (Vpr-VLPs) were used as negative controls. After VLP treatment, cells were collected and SAMHD1 immunoblots performed. Vinculin served as loading control. Endogenous SAMHD1 in WT cells and stably expressed GFP-SAMHD1 were degraded by Vpx. In contrast, SAMHD1-GFP was protected from Vpx degradation likely due to steric hindrance. Experiment performed only one time.

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