WT SAMHD1 relocates cytoplasm-localized mutants of SAMHD1K11A to the nucleus and facilitates their Vpx-induced degradation.

<p>(A)–(C) Wild-type SAMHD1-HA partially translocated pmCherry-SAMHD1 K11A to the nucleus. pmCherry-SAMHD1-HA WT and/or K11A with pEYFP-Nuc were co-transfected into HEK293T cells using PEI Max. Live cells were imaged at 24 h post-transfection. (D) Interaction between wild-type SAMHD1 and K11A. pSAMHD1 WT-Flag was co-transfected with pmCherry-SAMHD1-HA WT or K11A. Transfected cells were harvested after 48 h and incubated in lysis buffer for 30 min. Cell lysates were added to an anti-HA affinity matrix (Roche). Eluted protein samples were detected by immunoblotting with anti-HA and anti-Flag antibody to detect mCherry-SAMHD1 and SAMHD1-Flag, respectively. (E) Immunoblotting demonstrates that pmCherry-SAMHD1 K11A became sensitive to Vpx-mediated degradation when co-expressed with SAMHD1 WT. HEK293T cells were co-transfected with HA-Vpx and pmCherrySAMHD1 K11A and/or pSAMHD1-Flag wild-type. Cell extracts were harvested 48 h later and analyzed by SDS-PAGE, followed by immunoblotting to detect SAMHD1-HA and HA-Vpx. β-actin was used as the loading control.