Lentivirus mediated knockdown of Piccolo with interference RNAs. (A) Schematic diagram of Piccolo, illustrating its multidomain structure (a polyglutamate [pQ] region, two double zinc finger domains [Zn1/2], three coiled-coil domains [CC1-3], a PDZ domain, and two C2 domains [C2A/B]) and the location and sequence of the shRNA (Pclo28) used in this study. (B) Schematic diagram of the lentiviral vector (FUGW H1) used to express shRNAs under control of the H1 promoter (H1 prom; yellow) and either EGFP or EGFP-Synapsin1a (Syn1a) under the ubiquitin-C promoter. (C) Western blots of cellular lysates from hippocampal neurons (uninfected or infected with LV/EGFP-Synapsin1a [EGFP-Syn] or LV/EGFP-Synapsin1a/Pclo28 [EGFPSynPclo28]) immunostained for Piccolo, Bassoon, tubulin, Synaptophysin (Syph), and GFP-Synapsin (GFP-Syn). Protein molecular weights are indicated on the right. (D and E) Hippocampal neurons infected with LV/EGFP-Synapsin1a (EGFPSyn) or LV/EGFP-Synapsin1a/Pclo28 (EGFPSynPclo28) and immunostained with antibodies against MAP2 (Merged, blue) and Piccolo (D, red) or Bassoon (E, red) after 14 DIV. Only a fraction of presynaptic boutons express EGFP-Synapsin1a/Pclo28; Piccolo immunoreactivity is from uninfected neurons. Bars, 10 μm. (F and G) Bar graphs showing percent colocalization of EGFP-Synapsin1a (EGFPSyn) puncta with either Piccolo (F) or Bassoon (G) puncta along dendritic profiles (n > 2,000 puncta per condition, five experiments). Error bars indicate SEM.