anti-bioid2  (BioFront Technologies Inc)

Journal: STAR Protocols

Article Title: Protocol to study secretome interactions using extracellular proximity labeling

doi: 10.1016/j.xpro.2024.103509

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Article Snippet: A. aeolicus BPL/BioID2 antibody (1:2,000 dilution) , Novus Biologicals , NBP2-59941.

Techniques: Recombinant, Modification, Saline, Electron Microscopy, Protease Inhibitor, Mass Spectrometry, Expressing, Plasmid Preparation, Control, Software, Imaging, Spectrophotometry, Suction Filtration, Pore Size, Membrane

Journal: Nature Communications

Article Title: ARMH3 is an ARL5 effector that promotes PI4KB-catalyzed PI4P synthesis at the trans -Golgi network

doi: 10.1038/s41467-024-54410-y

Figure Lengend Snippet: a Immunofluorescence microscopy of WT and KO HeLa cells stained for endogenous PI4P (green) and the Golgi protein giantin (magenta). Single-channel images are shown in grayscale with nuclei (DAPI) in blue. Scale bars: 10 μm. Notice the disappearance of PI4P from the Golgi complex and the persistence of vesicular PI4P in all the KO cells. b Quantification of the Pearson’s correlation coefficient (PCC) of PI4P vs giantin from three independent, color-coded experiments such as that shown in panel a. c Quantification of PI4P intensity at the Golgi complex relative to the cytosol from experiments such as that shown in panel a. Data are represented as SuperPlots showing the mean ± SD of the means of the individual data points from three independent, color-coded experiments. The statistical significance of the differences was calculated by one-way ANOVA with multiple comparisons using Dunnett’s test. P- values are indicated on the graphs. Source data are provided in the Source Data file.

Article Snippet: The following primary antibodies (supplier, catalog number, working dilution) were used for immunoblotting and/or immunofluorescence microscopy: chicken anti-BioID2 (BioFront Technologies, BID2-CP-100, 1:1000 for IF), mouse HRP-conjugated anti-GFP (Miltenyi Biotec, 130-091-833, 1:2000 for WB), rat anti-HA epitope (Roche, 12158167001, 1:1000 for IF, 1:5000 for WB), rabbit anti-GFP (Invitrogen, A-11122, 1:1000 for IF), mouse anti-PI4KB (BD Biosciences, 611817, 1:200 for IF, 1:1000 for WB), mouse HRP-conjugated anti-α-tubulin (Santa Cruz Biotechnology, sc-32293 HRP, 1:5000 for WB), mouse anti-PI4P (Echelon Biosciences, Z-P004, 1:200 for IF), rabbit anti-giantin (Abcam, ab80864, 1:500 for IF), rabbit anti-ARMH3 (Invitrogen, PA5-62264, 1:200 for WB), rabbit anti-GOLPH3 (Abcam, ab98023, 1:200 for IF, 1:1000 for WB), mouse anti-CI-MPR (Abcam, ab2733, 1:200 for IF), sheep anti-TGN46 (Bio-Rad, AHP500G, 1:200 for IF, 1:5000 for WB), mouse anti-γ1-adaptin (BD Biosciences, 610385, 1:200 for IF), rabbit anti-β-COP (Invitrogen, PA1-061, 1:200 for IF), mouse anti-GGA3 (BD Biosciences, 612310, 1:200 for IF), mouse anti-ACBD3 (Sigma-Aldrich, WH0064746M1, 1:200 for IF), goat anti-ST6GAL1 (R&D Systems, AF5924, 1:200 for IF), mouse anti-LAMP1 to detect fully glycosylated protein (DSHB, H4A3-C, 1:5000 for WB) (Fig. ), rabbit anti-LAMP1 to detect both glycosylated and deglycosylated protein (Cell Signaling Technology, 9091, 1:1000 for WB) (Fig. ), Alexa Fluor 555 goat anti-chicken IgY (Invitrogen, A-21437, 1:1000 for IF), Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, A-21206, 1:1000 for IF), Alexa Fluor 555 donkey anti-mouse IgG (Invitrogen, A-31570, 1:1000 for IF), Alexa Fluor 647 donkey anti-rat IgG (Invitrogen, A-48272, 1:1000 for IF), Alexa Fluor 488 donkey anti-mouse IgG (Invitrogen, A-21202, 1:1000 for IF), Alexa Fluor 546 donkey anti-rabbit IgG (Invitrogen, A-10040, 1:1000 for IF).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: Research Square

Article Title: A protein-proximity screen reveals Ebola virus co-opts the mRNA decapping complex through the scaffold protein EDC4

doi: 10.21203/rs.3.rs-3838220/v1

Figure Lengend Snippet: a Top schematic shows arrangement of EBOV structural proteins in virion and encoding genes in the virus genome. Bottom shows constructs used in screening with placement of the BioID2 ligase indicated as green blocks. b Expression of viral protein-BioID2 fusion constructs and GFP-BioID2 non-specific control from stable cell line lysates. Blots were probed with biotin ligase specific antibody and loading levels controlled by detection of β-actin. c Blot of biotinylation levels, measured in cell lysates after inducing expression of each indicated protein by tetracycline and then adding biotin to cell medium to measure ligase activity. GFP-BioID2 was used as non-specific biotinylation control (second to last lane) as well as endogenous biotinylation in Flp-In TREx 293 cells not expressing any BioID2 construct (last lane). One representative experimental replicate of each construct is shown. d Hub and spoke network model showing identified interactions from screening. 447 host-viral protein interactions were identified between both N- and C- terminal constructs.

Article Snippet: Chicken anti-BioID2 primary antibody was purchased from BioFront Technologies (BioBID-CP-100).

Techniques: Virus, Construct, Expressing, Control, Stable Transfection, Activity Assay

Journal: Research Square

Article Title: A protein-proximity screen reveals Ebola virus co-opts the mRNA decapping complex through the scaffold protein EDC4

doi: 10.21203/rs.3.rs-3838220/v1

Figure Lengend Snippet: Each BioID2 screen was performed in quadruplicate. The top left shows endogenous biotinylation in the Flp-In TREx 293 cell line, which expresses no BioID2 construct, and the following panel represents non-specific biotinylation by the GFP-BioID2 construct. Subsequent panels demonstrate biotinylation of the indicated constructs. Following processing, samples were transferred to nitrocellulose membranes and stained with fluorescently labeled streptavidin.

Article Snippet: Chicken anti-BioID2 primary antibody was purchased from BioFront Technologies (BioBID-CP-100).

Techniques: Construct, Staining, Labeling

Journal: Research Square

Article Title: A protein-proximity screen reveals Ebola virus co-opts the mRNA decapping complex through the scaffold protein EDC4

doi: 10.21203/rs.3.rs-3838220/v1

Figure Lengend Snippet: PCSF was used to inter-relate BioID hits with the HIPPIE human protein-protein interaction (PPI) network. a The algorithm seeks to connect hits via the shortest pathways defined by the PPI network and considers confidence of underlying interactions and the presence of other hits along the mapped path. Hits are represented by nodes (green circles), where node size corresponds to the BioID hit score. At left, two paths are identified to connect strongly scored hits 1 and 4. The red path is the shortest to make the connection but requires inclusion of two host proteins that were not hits (Steiner nodes, triangles). The blue route is longer but has higher confidence, being better supported by literature citations, incorporates other hits from the screen and only one Steiner node. The algorithm will have preference for incorporating the blue route into the final network solution at right. b Overview of complete network mapping and gene set enrichment process. (Left) indicates how virus protein interactions from BioID screening were rescored as a portion of total SAINT scores for an associated host protein interaction. If a host protein was associated with multiple virus proteins it was labeled by a segmented circle using colors indicated in c (Right) to perform functional enrichment, subnetwork clusters were segregated by edge-betweenness measurements (red and purple dashed circles indicate clusters). Gene set enrichment was then performed for each cluster (red and purple circles). c Full network generated by PCSF for identified BioID2 screening hits related by the HIPPIE PPI. Five proteins of the mRNA decapping complex, each bound by VP35, are indicated in the inset. d Gene ontology annotated network obtained by Enrichr. Each color and corresponding number designate a different subnetwork with a functional enrichment listed in Extended data table 2.

Article Snippet: Chicken anti-BioID2 primary antibody was purchased from BioFront Technologies (BioBID-CP-100).

Techniques: Virus, Labeling, Functional Assay, Generated

Journal: The Journal of Biological Chemistry

Article Title: Comprehensive analysis of the proximity-dependent nuclear interactome for the oncoprotein NOTCH1 in live cells

doi: 10.1016/j.jbc.2023.105522

Figure Lengend Snippet: Identification of Notch1 proximity-dependent interacting proteins in HEK293 cells. A , the schematic of doxycycline-inducible constructs under the tetracycline response element (TRE) promoter. Notch1 intracellular domain (NICD) was fused to C terminus of the promiscuous biotin ligase BioID2. B , immunofluorescence analyses of HEK293 cells stably expressing fusion protein detected with anti-BioID2 ( red ) and promiscuous biotinylation detected with fluorescently labeled streptavidin ( green ) following the addition of exogenous doxycycline and biotin. The scale bar represents 20 μm. C , Western blot (WB) analysis of biotinylated proteins detected with streptavidin-HRP. Asterisks indicate the location of the BioID2-fusion protein (detected with anti-BioID2). Each lane represents a single biological replicate used in the BioID study. D , word cloud diagram depicting Notch1 interacting proteins identified by mass spectrometry (MS). The size of each protein name is proportional to the number of identified total MS/MS spectral counts for each protein. Candidate proteins previously reported listed as Notch1 interactors in the BioGRID database are shown in red , and previously unreported candidate Notch1 interactors are shown in blue . BioID, biotin identification; HRP, horseradish peroxidase.

Article Snippet: After blocking with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, the membrane was incubated with chicken anti-BioID2 primary antibody (1:5000; BID2-CP-100; BioFront Technologies).

Techniques: Construct, Immunofluorescence, Stable Transfection, Expressing, Labeling, Western Blot, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Comprehensive analysis of the proximity-dependent nuclear interactome for the oncoprotein NOTCH1 in live cells

doi: 10.1016/j.jbc.2023.105522

Figure Lengend Snippet: Identification of Notch1 proximity-dependent interacting proteins in 3T3 cells. A , schematic of doxycycline-inducible constructs under the tetracycline response element (TRE) promoter for BioID2-only and BioID2-NICD. B , immunofluorescence analyses of 3T3 cells stably expressing fusion protein detected with anti-BioID2 ( red ) and promiscuous biotinylation detected with fluorescently labeled streptavidin ( green ) following the addition of exogenous doxycycline and biotin. The scale bar represents 20 μm. C , Western blot (WB) analysis of biotinylated proteins detected with streptavidin-HRP. Asterisks indicate the location of the BioID2-fusion protein (detected with anti-BioID2). Each lane represents a single biological replicate used in the BioID study. D , word cloud diagram depicting Notch1 interacting proteins identified by mass spectrometry (MS). The size of each protein name is proportional to the number of identified total MS/MS spectral counts for each protein. Candidate proteins previously reported listed as Notch1 interactors in the BioGRID database are shown in red , and previously unreported candidate Notch1 interactors are shown in blue . BioID, biotin identification; HRP, horseradish peroxidase.

Article Snippet: After blocking with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, the membrane was incubated with chicken anti-BioID2 primary antibody (1:5000; BID2-CP-100; BioFront Technologies).

Techniques: Construct, Immunofluorescence, Stable Transfection, Expressing, Labeling, Western Blot, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Comprehensive analysis of the proximity-dependent nuclear interactome for the oncoprotein NOTCH1 in live cells

doi: 10.1016/j.jbc.2023.105522

Figure Lengend Snippet: Identification of Notch1 proximity-dependent interacting proteins in HEK293 cells. A , the schematic of doxycycline-inducible constructs under the tetracycline response element (TRE) promoter. Notch1 intracellular domain (NICD) was fused to C terminus of the promiscuous biotin ligase BioID2. B , immunofluorescence analyses of HEK293 cells stably expressing fusion protein detected with anti-BioID2 ( red ) and promiscuous biotinylation detected with fluorescently labeled streptavidin ( green ) following the addition of exogenous doxycycline and biotin. The scale bar represents 20 μm. C , Western blot (WB) analysis of biotinylated proteins detected with streptavidin-HRP. Asterisks indicate the location of the BioID2-fusion protein (detected with anti-BioID2). Each lane represents a single biological replicate used in the BioID study. D , word cloud diagram depicting Notch1 interacting proteins identified by mass spectrometry (MS). The size of each protein name is proportional to the number of identified total MS/MS spectral counts for each protein. Candidate proteins previously reported listed as Notch1 interactors in the BioGRID database are shown in red , and previously unreported candidate Notch1 interactors are shown in blue . BioID, biotin identification; HRP, horseradish peroxidase.

Article Snippet: Cells grown on 1.5 mm glass coverslips were fixed with 3% (w/v) paraformaldehyde/phosphate-buffered saline for 10 min and permeabilized with 0.4% (w/v) Triton X −100/PBS for 15 min. To detect BioID2 fusion proteins, chicken anti-BioID2 (1:5000; BID2-CP-100; BioFront Technologies) was used ( ).

Techniques: Construct, Immunofluorescence, Stable Transfection, Expressing, Labeling, Western Blot, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Comprehensive analysis of the proximity-dependent nuclear interactome for the oncoprotein NOTCH1 in live cells

doi: 10.1016/j.jbc.2023.105522

Figure Lengend Snippet: Identification of Notch1 proximity-dependent interacting proteins in 3T3 cells. A , schematic of doxycycline-inducible constructs under the tetracycline response element (TRE) promoter for BioID2-only and BioID2-NICD. B , immunofluorescence analyses of 3T3 cells stably expressing fusion protein detected with anti-BioID2 ( red ) and promiscuous biotinylation detected with fluorescently labeled streptavidin ( green ) following the addition of exogenous doxycycline and biotin. The scale bar represents 20 μm. C , Western blot (WB) analysis of biotinylated proteins detected with streptavidin-HRP. Asterisks indicate the location of the BioID2-fusion protein (detected with anti-BioID2). Each lane represents a single biological replicate used in the BioID study. D , word cloud diagram depicting Notch1 interacting proteins identified by mass spectrometry (MS). The size of each protein name is proportional to the number of identified total MS/MS spectral counts for each protein. Candidate proteins previously reported listed as Notch1 interactors in the BioGRID database are shown in red , and previously unreported candidate Notch1 interactors are shown in blue . BioID, biotin identification; HRP, horseradish peroxidase.

Article Snippet: Cells grown on 1.5 mm glass coverslips were fixed with 3% (w/v) paraformaldehyde/phosphate-buffered saline for 10 min and permeabilized with 0.4% (w/v) Triton X −100/PBS for 15 min. To detect BioID2 fusion proteins, chicken anti-BioID2 (1:5000; BID2-CP-100; BioFront Technologies) was used ( ).

Techniques: Construct, Immunofluorescence, Stable Transfection, Expressing, Labeling, Western Blot, Mass Spectrometry, Tandem Mass Spectroscopy