Transient Nedd4-binding protein 3 knockdown results in impaired neurite branching. (A) Characterization of the Nedd4-binding protein 3 (N4BP3) interfering RNA (RNAi) construct. HEK-293T cells were cotransfected with Myc-N4BP3 and either the empty pSUPER control vector (Oligoengine, Seattle, WA, USA) or the N4BP3 RNAi construct. Western blot of the corresponding cell lysates shows reduction of Myc-N4BP3 in the presence of N4BP3 RNAi with either N4BP3 or Myc antibodies as indicated. (B) All culture wells used for transient transfection experiments were immunostained with a phosphorylated inhibitor of κB, subunit α (p-IκBα) antibody (Alexa Fluor 568, red; left panel) to delineate the axon initial segment (AIS; filled arrowheads) to distinguish between axon (filled arrowheads) and dendrites (framed arrowheads). GFP, green fluorescent protein. (C) Representative images of primary rat hippocampal neurons transiently transfected (DIV3 + 2) with either the empty pSUPER control vector or the N4BP3 RNAi construct as indicated. Statistical evaluation of axon length (D); the number of primary, secondary and tertiary branches (E); the axon complexity index (ACI) (F); and the (primary) branches per 100-μm axonal length (G) of pSUPER control vector (black bars) vs. N4BP3 RNAi (gray bars) transfected neurons. (H) Representative images of rat hippocampal neurons transiently transfected (DIV8 + 3) with either the empty pSUPER control vector or the N4BP3 RNAi construct as indicated. Statistical evaluation of the total number of dendritic end tips (I) and Sholl analysis (J) of pSUPER control vector (black bar in (I), squares in (J)) vs. N4BP3 RNAi (gray bar in (I), gray squares in (J)) transfected neurons. n = 10 cells from three independent experiments in (D), (E), (F), (G), (I) and (J) (Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001).