Fig. 3.

Protein-DNA binding assays showing (A) NPM1 interaction with GQRS structure within c-myc (myc, 1st lane) and AAV ITR sequences (ITRA, 2nd lane) in 293T cells after transfection with AAV packaging plasmids and a trace NPM1 interaction with c-myc (myc, 4th lane) and ITR (ITRA, 5th lane) GQRS in NPM1 knockdown cells after AAV plasmid transfection; a non-GQRS DNA oligonucleotide (NGQRS) was included as nonspecific DNA binding control (NGQRS, 6th lane); (B) Competition for AAV_ITR binding to NPM1 proteins using an increased amount of c-myc GQRS (top and middle panels): a progressively diminishing of ITR_A (top panel) or ITR_B GQRS (middle panel) binding to NPM1 proteins; a non-GQRS DNA oligonucleotide (NGQRS, bottom panel) was also used as assay control, showing that non-GQRS DNA oligonucleotides (NGQRS) cannot compete out the AAV ITR_A GQRS binding to NPM1 proteins (bottom panel) and that AAV ITR binding to NPM1 proteins is GQRS specific; (C) NPM1 expression in 293T and NPM1 knockdown cells before DNA binding assays. A non-GQRS DNA oligonucleotide (NGQRS) was included as nonspecific DNA binding control and Housekeeping gene GAPDH was used as a loading control.