Journal: bioRxiv

Article Title: Cell-Surface Proteomic Profiling in the Fly Brain Uncovers New Wiring Regulators

doi: 10.1101/819037

Figure Lengend Snippet: ( A ) Scheme and features of cell-surface biotinylation in intact tissues. ( B ) Olfactory projection neuron (PN) specific VT033006-GAL4 ( PN-GAL4 , hereafter) drove the expression of membrane-targeted GFP (CD4-GFP). The zoom-in panel shows a single optical section of the PN soma area. Orange circle, antennal lobe. ( C and D ) Neutravidin staining of antennal lobes after the cell-surface biotinylation reaction. (C) HRP was not expressed by omitting the GAL4 driver. (D) PN-GAL4 drove the expression of cell surface-targeted HRP (HRP-CD2). The zoom-in panel shows a single optical section of the PN soma area. ( E ) Left and middle, streptavidin blots of the whole-brain lysate (left) and the post-enrichment bead eluate (middle). Right, silver stain of the post-enrichment bead eluate (right). –HRP , PN-GAL4 omitted; +HRP , PN>HRP-CD2 . ( F ) Immunoblots of intracellular proteins, actin and bruchpilot (Brp), and neuronal surface protein N-cadherin (NCad) in the whole-brain lysate and the post-enrichment bead eluate. –HRP , PN-GAL4 omitted; +HRP , PN>HRP-CD2 . Scale bar, 10 μm. See also .

Article Snippet: Primary antibodies used in immunostaining include: rat anti-NCad (1:40; DN-Ex#8, Developmental Studies Hybridoma Bank), chicken anti-GFP (1:1000; GFP-1020, Aves Labs), rabbit anti-DsRed (1:200; 632496, Clontech), mouse anti-rat CD2 (1:200; OX-34, Bio-Rad), mouse anti-FLAG (1:100; M2, Sigma-Aldrich), mouse anti-V5 (1:100; R960-25, Thermo Fisher), and rabbit anti-LRP1 (1:200; gift of Suzanne Eaton) ( ).

Techniques: Expressing, Staining, Silver Staining, Western Blot

Journal: bioRxiv

Article Title: Cell-Surface Proteomic Profiling in the Fly Brain Uncovers New Wiring Regulators

doi: 10.1101/819037

Figure Lengend Snippet: ( A ) Scheme of the fly olfactory circuit. ( B and C ) Neutravidin staining of PN axon-targeted brain regions after the cell-surface biotinylation reaction. (B) HRP was not expressed by omitting the GAL4 driver. (C) PN-GAL4 drove the expression of cell surface-targeted HRP (HRP-CD2). Scale bar, 10 μm.

Article Snippet: Primary antibodies used in immunostaining include: rat anti-NCad (1:40; DN-Ex#8, Developmental Studies Hybridoma Bank), chicken anti-GFP (1:1000; GFP-1020, Aves Labs), rabbit anti-DsRed (1:200; 632496, Clontech), mouse anti-rat CD2 (1:200; OX-34, Bio-Rad), mouse anti-FLAG (1:100; M2, Sigma-Aldrich), mouse anti-V5 (1:100; R960-25, Thermo Fisher), and rabbit anti-LRP1 (1:200; gift of Suzanne Eaton) ( ).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Cell-Surface Proteomic Profiling in the Fly Brain Uncovers New Wiring Regulators

doi: 10.1101/819037

Figure Lengend Snippet: ( A ) Selection criteria for the genetic screen. Screen zone cutoffs: log 2 (mature/developing FC) < – 0.4 and –log 10 ( p value) > 1. FC, fold change. ( B ) Scheme and features of the genetic screen in ventromedial (VM) PNs and ORNs. VM5d and VM5v PNs were labelled by GMR86C10-LexA -driven membrane-targeted tdTomato ( LexAop-mtdTomato ; red). VM5v and VA2 ORNs were labelled by Or98a promoter-driven membrane-targeted GFP ( Or98a-mCD8-GFP ; cyan) and Or92a promoter-driven rat CD2 transmembrane motif ( Or92a-rCD2 ; magenta), respectively. The pan-neuronal C155-GAL4 drove the expression of gene-specific RNAi. ( C ) Targeting of VM5d/v PN dendrites and VM5v/VA2 ORN axons in a control antennal lobe. None of the 62 examined antennal lobes in controls exhibited any targeting defects. Dashed circle, antennal lobe; asterisk, PN soma. ( D ) CG6821/Lsp1γ knockdown caused global disruption of the antennal lobe structure. Yellow dashed circles in neuropil staining (blue) represent stereotyped glomeruli that are easily identified in control but misshapen and unrecognizable in Lsp1γ knockdown. ( E ) CG31998 and CG17839 knockdown caused long-range mistargeting of ORN axons and PN dendrites, respectively, to the dorsolateral (DL) antennal lobe. ( F ) CG33087/LRP1 and CG9796/GILT1 knockdown caused VM local mistargeting of ORN axons and PN dendrites. ( G ) Hit rates of our previous molecular family-based screen and the current cell-surface proteome guided screen, with the identical assay (scheme in B). Scale bar, 10 μm. D, dorsal; L, lateral. RNAi phenotypic penetrances are listed in . See also .

Article Snippet: Primary antibodies used in immunostaining include: rat anti-NCad (1:40; DN-Ex#8, Developmental Studies Hybridoma Bank), chicken anti-GFP (1:1000; GFP-1020, Aves Labs), rabbit anti-DsRed (1:200; 632496, Clontech), mouse anti-rat CD2 (1:200; OX-34, Bio-Rad), mouse anti-FLAG (1:100; M2, Sigma-Aldrich), mouse anti-V5 (1:100; R960-25, Thermo Fisher), and rabbit anti-LRP1 (1:200; gift of Suzanne Eaton) ( ).

Techniques: Selection, Expressing, Staining