Figure 1. Proteomic identification of... | Rockefeller University Press

Figure 1.

$Figure 1. Proteomic identification of Farp1, a synaptic protein reduced in SynCAM 1 KO mice. (A) Organization of Farp1 into FERM, DH, and PH domains. DH-PH domains are characteristic of GEFs. Black rectangle, epitope detected by the antibody. (B) Quantitative immunoblotting of SynCAM 1 KO hippocampi confirms Farp1 reduction compared with WT mice. Equal protein amounts were loaded. For quantification described in the text, signals were normalized to the loading control VCP. (C) Farp1 is enriched in brain determined by immunoblotting of equal protein amounts of adult rat tissues. Synaptotagmin 1 and actin were loading controls. (D) Farp1 expression throughout brain shown by immunoblotting of equal protein amounts. (E) Fractionation of P13 rat forebrain. Farp1 enriches in synaptic plasma membranes (SPM), similar to SynCAM 1. Farp1 in the crude synaptic vesicle (SV) preparation is presumably caused by nonvesicular content (Fogel et al., 2007). PNS, postnuclear supernatant; syn. sup., synaptosomal supernatant; synapt., synaptosomes. (F) Synaptosomal extracts prepared at P16 by sequential detergent extraction contain Farp1. A fraction is present in PSDs. PSD-95 served as a positive control and CASK and synaptophysin served as negative controls. (G) Farp1 localizes to dendritic protrusions. Confocal image of dissociated hippocampal neurons expressing GFP-Farp1 (green) at 12 div. Colocalization with the F-actin probe UtrCH-Cherry (red) and postsynaptic Shank (blue) is marked by circles. Single channels from the boxed area are enlarged below. Bars: (top) 5 µm; (insets) 2.5 µm.$

Proteomic identification of Farp1, a synaptic protein reduced in SynCAM 1 KO mice. (A) Organization of Farp1 into FERM, DH, and PH domains. DH-PH domains are characteristic of GEFs. Black rectangle, epitope detected by the antibody. (B) Quantitative immunoblotting of SynCAM 1 KO hippocampi confirms Farp1 reduction compared with WT mice. Equal protein amounts were loaded. For quantification described in the text, signals were normalized to the loading control VCP. (C) Farp1 is enriched in brain determined by immunoblotting of equal protein amounts of adult rat tissues. Synaptotagmin 1 and actin were loading controls. (D) Farp1 expression throughout brain shown by immunoblotting of equal protein amounts. (E) Fractionation of P13 rat forebrain. Farp1 enriches in synaptic plasma membranes (SPM), similar to SynCAM 1. Farp1 in the crude synaptic vesicle (SV) preparation is presumably caused by nonvesicular content (Fogel et al., 2007). PNS, postnuclear supernatant; syn. sup., synaptosomal supernatant; synapt., synaptosomes. (F) Synaptosomal extracts prepared at P16 by sequential detergent extraction contain Farp1. A fraction is present in PSDs. PSD-95 served as a positive control and CASK and synaptophysin served as negative controls. (G) Farp1 localizes to dendritic protrusions. Confocal image of dissociated hippocampal neurons expressing GFP-Farp1 (green) at 12 div. Colocalization with the F-actin probe UtrCH-Cherry (red) and postsynaptic Shank (blue) is marked by circles. Single channels from the boxed area are enlarged below. Bars: (top) 5 µm; (insets) 2.5 µm.