Localization of Fer at excitatory synapses and delocalization of synaptic vesicle and CAZ puncta after knockdown of Fer. (A and B) 14 DIV dissociated rat hippocampal neurons were stained with anti-Fer and anti-vGlut1 (A) or anti–PSD-95 (B) antibodies. (C) Localization of synaptophysin-GFP in 14 DIV hippocampal neurons that were transfected at 10 DIV with synaptophysin-GFP together with the indicated constructs. (D) Localization of GFP–Bsn95-3938 in 14 DIV hippocampal neurons transfected at 10 DIV with GFP–Bsn95-3938 plus control or Fer shRNA vector. Cultures were stained with anti-vGlut1 antibody. (E) 10 DIV neurons were transfected with GFP-containing vector or Fer shRNA and stained with Bassoon antibody. (left) Images were processed to indicate that Bassoon colocalized with GFP. (F and G) The coverage (normalized micrometers of GFP per 10 μm of axon length; see Materials and methods) of synaptophysin-GFP (Syn-GFP) puncta along axons in each indicated condition. In F, n = 7 (vector), 11 (Fer shRNA), 15 (Fer shRNA + WTFer*), and 13 (Fer shRNA + kinase-dead mutant [K591R] of Fer [KDFer*]). In G, n = 10 (vector), 9 (WT Fer), and 9 (kinase-dead mutant [K591R] of Fer). (H) The axonal coverage of GFP–Bsn-95-3938. n = 12 (vector) and 13 (Fer shRNA). (I and J) The cumulative percentage curves representing the feret length of synaptophysin-GFP (I) and endogenous Bassoon (J). In I, n = 281 (vector) and 297 (Fer shRNA). In J, n = 425 (vector) and 572 (Fer shRNA). WTFer* and KDFer* are shRNA refractory constructs. Boxed areas in A–C are shown in high resolution images at the right (A and B) or bottom (C) of each panel. Arrows and arrowheads indicate vGlut1 or Bassoon immunoreactivity along the transfected axons. *, P < 0.05; **, P < 0.01. Error bars represent SEM. Bars, 5 μm.