Fig 5. NDRG1 directly interacts with PCNA.

(A)Schematic procedure for purification and identification of NDRG1 binding proteins via TAP assay (left). Plasmid expressing Strep-Flag-tagged NDRG1 was stable transfected into KSHV positive iSLK.RGB cells. The equivalent empty vector was stable transfected as a control. Cell lysates were subjected to affinity purification with streptavidin beads, followed by IP with flag M2 beads. The purified elutes were resolved by SDS-PAGE and visualized with silver staining (right), and were also analyzed by MS. (B) Pathway pie chart showing classified and predicted functions of MS identified proteins. (C) Co-IP of NDRG1 and PCNA in HEK293T cells. Strep-Flag-tagged NDRG1 was transfected into cells along with pCDNA3.1-PCNA or empty vector controls. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-NDRG1 and anti-PCNA antibodies. (D) In vitro interaction between NDRG1 and PCNA via GST pull down assay. Purified GST, and GST-fused full length NDRG1 were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent in vitro translated IVT–PCNA, and pulled down proteins were subjected to western blotting detection (upper panel). (E) NDRG1 colocalized with PCNA in the nucleus. Cells were fixed and probed with rabbit antibody against NDRG1 and mouse antibody against PCNA, followed by incubation with goat anti-rabbit IgG conjugated with Alexa Fluor 555 (red), goat anti-mouse IgG conjugated with Alexa Fluor 488 (green), DAPI (blue). Scale bars represent 5μm.