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

Journal: Genes

Article Title: Establishment and Expression of Cytokines in a Theileria annulata -Infected Bovine B Cell Line

doi: 10.3390/genes10050329

Figure Lengend Snippet: Percentage of CD21 + cells in peripheral blood mononuclear cells (PBMCs) and analysis of their purity after magnetic separation. Here, ( A ) is the background control, ( B ) is the percentage of CD21 + cells in PBMCs, and ( C ) is the purity of magnetically isolated cells.

Article Snippet: The mouse anti-bovine CD21 (MCA1424PE), mouse anti-bovine IgM (AAI19F), mouse anti-bovine WC4 (MCA1648G), and negative control mouse IgG1 (MCA928F) antibodies were purchased from Bio-Rad (Hercules, CA, USA).

Techniques: Control, Isolation

Journal: Genes

Article Title: Establishment and Expression of Cytokines in a Theileria annulata -Infected Bovine B Cell Line

doi: 10.3390/genes10050329

Figure Lengend Snippet: Percentage of CD21 in transformed B cells at the 3rd passage.

Article Snippet: The mouse anti-bovine CD21 (MCA1424PE), mouse anti-bovine IgM (AAI19F), mouse anti-bovine WC4 (MCA1648G), and negative control mouse IgG1 (MCA928F) antibodies were purchased from Bio-Rad (Hercules, CA, USA).

Techniques: Transformation Assay

Journal: Genes

Article Title: Establishment and Expression of Cytokines in a Theileria annulata -Infected Bovine B Cell Line

doi: 10.3390/genes10050329

Figure Lengend Snippet: Percentages of B cell-specific surface markers (CD21, IgM, and WC4) present in transformed cells (6th and 10th generations), single-cell line clones (approximately 15th generation), and normal purified cells. The percentages of CD21 (98.8, 99.7, 98.0, 98.5, and 97%; mean ± SD of 98.4 ± 1.00), IgM (3.51, 0.64, 1.42, 0.99, and 2.83%; mean ± SD of 1.88 ± 1.23) and WC4 (0.51, 1.96, 0.71, 0.80, and 0.97%; mean ± SD of 0.99 ± 0.57) in five clones were determined. CD21 was present in mono- and polyclonal cell lines, while negligible percentage of IgM and WC4 were present.

Article Snippet: The mouse anti-bovine CD21 (MCA1424PE), mouse anti-bovine IgM (AAI19F), mouse anti-bovine WC4 (MCA1648G), and negative control mouse IgG1 (MCA928F) antibodies were purchased from Bio-Rad (Hercules, CA, USA).

Techniques: Transformation Assay, Clone Assay, Purification

Journal: Vaccines

Article Title: Antigen Targeting of Porcine Skin DEC205 + Dendritic Cells.

doi: 10.3390/vaccines10050684

Figure Lengend Snippet: Figure 1. Design, expression, and characterization of rAb ZH9F7-Cap and isotype control 7DEU-Cap. (a) Schematic illustration of the recombinant mouse × pig chimeric rAb ZH9F7-Cap and isotype control rAb 7DEU-Cap. (b) 10% SDS–PAGE followed by Coomassie blue staining of purified rAb ZH9F7-Cap and rAb 7DEU-Cap. Lane 1: molecular weight marker; lane 2: rAb ZH9F7 without Cap; lane 3: purified rAb ZH9F7-Cap; lane 4: purified rAb 7DEU-Cap. (c) Western blot of the chimeric mouse × pig rAb ZH9F7-Cap and rAb 7DEU-Cap. Lane 1: molecular weight marker; lane 2: rAb ZH9F7 without Cap; lane 3: purified rAb ZH9F7-Cap; lane 4: purified rAb 7DEU-Cap. HRP- conjugated goat anti-porcine IgG (H + L) was used to confirm the expression of both mouse × pig recombinant antibodies.

Article Snippet: After Fc receptors were blocked, 3 × 106 cells were stained with IgG1 mouse antiCD3 (Southern Biotech, Birmingham, AL, USA), IgG1 mouse anti-CD21 (Southern Biotech, Birmingham, AL, USA), and IgY chicken anti-CADM1 (MBL International, Woburn, MA, USA) at room temperature for 15 min. Then, BV421-conjugated goat anti-mouse IgG1 (BioLegend, San Diego, CA, USA) and Alexa Fluor 647-conjugated goat anti-chicken IgY (Invitrogen, Waltham, MA, USA) were added and incubated at room temperature for 15 min.

Techniques: Expressing, Control, Recombinant, SDS Page, Staining, Molecular Weight, Marker, Western Blot

Journal: Vaccines

Article Title: Antigen Targeting of Porcine Skin DEC205 + Dendritic Cells.

doi: 10.3390/vaccines10050684

Figure Lengend Snippet: Figure 7. Immune response promoted by intradermal application of rAb ZH9F7-Cap in swine. (a) Immunization schedule. (b) Indirect ELISA for the detection of PCV2a anti-Cap antibodies. Serum samples were tested for anti-PCV2a Cap IgG detection at weeks 0 and 4 postimmunization. (c) Dot plots represent the intracellular IFN-γ in cells stimulated with PHA, rAb ZH9F7-Cap (d), and rAb ZH9F7 without PCV2 Cap as a control (e). In (b–d), graphs represent the average percentage of IFN-γ expression by the CD4+CD8−, CD4−CD8+, and CD4+CD8+ populations. Black bars-dots represent cells from the control group (n = 2), and blue bars-dots represent cells from the rAb ZH9F7-Cap vaccinated group (n = 3). Two-way ANOVA and Tukey–Kramer tests for multiple comparisons of means were performed (p < 0.05). p-values denote statistically significant differences in the means of the different treatments. In (b–d), the basal expression of IFN-γ from unstimulated cells was subtracted.

Article Snippet: After Fc receptors were blocked, 3 × 106 cells were stained with IgG1 mouse antiCD3 (Southern Biotech, Birmingham, AL, USA), IgG1 mouse anti-CD21 (Southern Biotech, Birmingham, AL, USA), and IgY chicken anti-CADM1 (MBL International, Woburn, MA, USA) at room temperature for 15 min. Then, BV421-conjugated goat anti-mouse IgG1 (BioLegend, San Diego, CA, USA) and Alexa Fluor 647-conjugated goat anti-chicken IgY (Invitrogen, Waltham, MA, USA) were added and incubated at room temperature for 15 min.

Techniques: Indirect ELISA, Control, Expressing