Journal: bioRxiv

Article Title: Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1)

doi: 10.1101/2023.02.09.527921

Figure Lengend Snippet: ( A ) Experimental design: BioID2-fusion constructs of wild-type (WT) or mutant (H543R/T545A) GPR133 were overexpressed in HEK293T cells. Following treatment with biotin, biotinylated proteins were purified using Neutravidin beads. Purified proteins were analyzed by mass spectrometry. ( B ) Top 30 biotinylated proteins with statistically equivalent detection in the two experimental conditions were ranked based on their mean MS intensity. ESYT1 (arrow) shows the highest intensity of all biotinylated proteins in close proximity to GPR133, independent on GPR133 cleavage and signaling. Gβ subunits are also identified (red box). ( C ) Structure and function of ESYT1. ( Ci ) Structural domains of ESYT1. ( Cii ) ESYT1 dimers form ER-PM tethers in response to elevations in cytosolic Ca 2+ . ( D ) Co-purification confirms binding of ESYT1 to TwinStrep-tagged GPR133, both WT and the uncleavable H543R mutant. ( Di ) Input samples: Whole cell lysates of HEK293T cells expressing wild-type GPR133 or the cleavage-deficient mutant GPR133 (H543R) with a C-terminal TwinStrep-tag following transfection with ESYT1. ( Dii ) Elution samples following Strep-Tactin purification. WB, Western blot; C-term, antibody against the cytosolic C-terminus of GPR133.

Article Snippet: Fusion constructs of wild-type GPR133-BioID2 and mutant GPR133 (H543R/T545A)-BioID2 were created using the MCS-13X Linker-BioID2-HA (addgene #80899) ( Kim et al ., 2016 ) plasmid and subsequently subcloned into the lentiviral backbone pLVX-EF1a-mCherry-N1 (Takara, Cat# 631986).

Techniques: Construct, Mutagenesis, Purification, Mass Spectrometry, Copurification, Binding Assay, Expressing, Transfection, Western Blot

Journal: bioRxiv

Article Title: Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1)

doi: 10.1101/2023.02.09.527921

Figure Lengend Snippet: ( A-D ) ESYT1 knockdown. ( A ) Western blot confirms reduced levels of endogenous ESYT1 following its knockdown (shESYT1) compared to the control (shSCR), and stable expression of GPR133, in transduced HEK293T cells. ( B ) GPR133 surface expression is not affected by ESYT1 knockdown in ELISA assays (two-way ANOVA, p>0.05). ns, not significant; A 450 nm , absorbance/optical density at 450 nm. Bars represent mean ± SEM of 3 experiments. ( C ) Immunofluorescent staining shows no change in the subcellular localization of GPR133 following knockdown of ESYT1 compared to the control. ( D ) Intracellular cAMP levels increase significantly in GPR133 expressing HEK293T cells after knockdown of ESYT1 compared to the control (two-way ANOVA F (1,8) =503.2, p<0.0001; Sidak’s post hoc test: GPR133 + shSCR vs. GPR133 + shESYT1, p<0.0001). Bars represent mean ± SEM of 3 experiments. ( E-H ) ESYT1 knockout. ( E ) Western blot confirms reduced levels of endogenous ESYT1 following the KO compared to the control (Rosa26). Expression of GPR133 is not affected by ESYT1 KO in transfected HEK293T cells. ( F ) GPR133 surface expression does not change upon KO of ESYT1 compared to the Rosa26 control in ELISA assays (two-way ANOVA, p>0.05). Bars represent mean ± SEM of 3 experiments. ns, not significant; A 450 nm , absorbance/optical density at 450 nm. ( G ) Immunofluorescent staining of HEK293T cells transfected with GPR133 shows no change in GPR133 subcellular localization in ESYT1 KO cells compared to the control cells (Rosa26). ( H ) Significant increase of cAMP concentrations in GPR133 expressing HEK293T cells following KO of ESYT1 compared to the control (two-way ANOVA F (1,8) =10.92, p=0.0108; Sidak’s post hoc test: GPR133 + Rosa26 vs. GPR133 + ESYT1 KO, p=0.0027). Bars represent mean ± SEM of 3 experiments.

Article Snippet: Fusion constructs of wild-type GPR133-BioID2 and mutant GPR133 (H543R/T545A)-BioID2 were created using the MCS-13X Linker-BioID2-HA (addgene #80899) ( Kim et al ., 2016 ) plasmid and subsequently subcloned into the lentiviral backbone pLVX-EF1a-mCherry-N1 (Takara, Cat# 631986).

Techniques: Knockdown, Western Blot, Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Knock-Out, Transfection

Journal: bioRxiv

Article Title: Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1)

doi: 10.1101/2023.02.09.527921

Figure Lengend Snippet: ( A-D ) ESYT1-GFP overexpression. ( A ) Western blot confirms increased ESYT1-GFP protein levels following transfection of GPR133 expressing cells. GPR133 expression levels are not affected in HEK293T cells. ( B ) GPR133 surface expression remains unchanged following overexpression of ESYT1-GFP (two-way ANOVA, p>0.05). Bars represent mean ± SEM of 4 experiments. ns, not significant; ns, not significant; A 450 nm , absorbance/optical density at 450 nm. ( C ) Immunofluorescent staining of HEK293T cells expressing GPR133 and ESYT1-GFP. The cellular distribution of GPR133 immunoreactivity is unchanged. ( D ) Intracellular cAMP levels significantly decrease in GPR133-expressing HEK293T cells following overexpression of ESYT1-GFP compared to the control (two-way ANOVA F (1,12) =7.928, p<0.0156; Sidak’s post hoc test: GPR133 + CTRL vs. GPR133 + ESYT1, p=0.0041). Bars represent mean ± SEM of 4 experiments. ns, not significant. ( E-F ) ESYT1 overexpression rescues the effect of ESYT1 knockdown in GPR133-overexpressing cells. ( E ) Western Blot confirming ESYT1 knockdown and overexpression in HEK293T cells and HEK293T cells overexpressing GPR133. Expression levels of GPR133 were not affected following knockdown or overexpression of ESYT1. ( F ) Intracellular cAMP levels of GPR133 expressing HEK293T cells are normalized to shSCR. Bars represent mean ± SEM of 4 experiments. Compared to the control (shSCR), GPR133 signaling increases significantly following transduction with shESYT1 and decreases significantly following transfection with ESYT1. ESYT1 overexpression rescues the increase in cAMP levels after ESYT1 KD (one-way ANOVA F (3,12) =24.64, p<0.0001; Tukey’s post hoc test: shSCR vs. shESYT1, p=0.0030; shSCR vs. shSCR + ESYT1, p=0.0094; shESYT1 vs. shSCR + ESYT1, p<0.0001; shESYT1 vs. shESYT1 + ESYT1, p=0.0217; shSCR + ESYT1 vs. shESYT1 + ESYT1, p=0.0014). Bars represent mean ± SEM of 4 experiments. ns, not significant.

Article Snippet: Fusion constructs of wild-type GPR133-BioID2 and mutant GPR133 (H543R/T545A)-BioID2 were created using the MCS-13X Linker-BioID2-HA (addgene #80899) ( Kim et al ., 2016 ) plasmid and subsequently subcloned into the lentiviral backbone pLVX-EF1a-mCherry-N1 (Takara, Cat# 631986).

Techniques: Over Expression, Western Blot, Transfection, Expressing, Staining, Control, Knockdown, Transduction

Journal: bioRxiv

Article Title: Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1)

doi: 10.1101/2023.02.09.527921

Figure Lengend Snippet: ( A ) Schematic showing ESYT1 deletion mutants used in this experiment. ( B ) GPR133 surface expression in ELISA assays following transfection of control HEK293T cells and HEK293T cells stably expressing GPR133 with different ESYT1 constructs. Overexpression of ESYT1, ΔC2C, ΔC2E or ΔC2C+E did not affect GPR133 surface expression compared to the vector control (two-way ANOVA, p>0.05). Bars represent mean ± SEM of 5 to 8 experiments. A 450 nm , absorbance/optical density at 450 nm. ( C ) Intracellular cAMP levels following transfection of HEK293T cells stably expressing GPR133 with different ESYT1 wild-type or mutant constructs. Concentrations of cAMP were significantly decreased in GPR133 expressing cells after transfection with ESYT1 and ΔC2E compared to the vector control. Overexpression of ΔC2C increased cAMP levels compared to the vector control and wild-type ESYT1 in GPR133-expressing HEK293T cells (two-way ANOVA F (4,46) =9.471, p<0.0001; Sidak’s post hoc test: GPR133 + vector vs. GPR133 + ESYT1, p=0.0001; GPR133 + vector vs. GPR133 + ΔC2C, p=0.0080; GPR133 + ESYT1 vs. GPR133 + ΔC2C, p<0.0001; GPR133 + ESYT1 vs. GPR133 + ΔC2C+E, p=0.0002; GPR133 + ΔC2E vs. GPR133 + ΔC2C+E, p=0.0218). Bars represent mean ± SEM of 5 to 8 experiments. ( D ) Affinity purification analysis testing binding of different ESYT1 constructs to GPR133. Input samples represent whole cell lysates of naïve HEK293T cells and HEK293T cells stably overexpressing GPR133 transfected with ESYT1 wild-type or deletion constructs. Elution samples following Strep-Tactin purification demonstrate that ESYT1 specific bands are only detected in GPR133 expressing cells transfected with wild-type ESYT1 and ΔC2E, but not after transfection with ΔC2C or ΔC2C+E.

Article Snippet: Fusion constructs of wild-type GPR133-BioID2 and mutant GPR133 (H543R/T545A)-BioID2 were created using the MCS-13X Linker-BioID2-HA (addgene #80899) ( Kim et al ., 2016 ) plasmid and subsequently subcloned into the lentiviral backbone pLVX-EF1a-mCherry-N1 (Takara, Cat# 631986).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Control, Stable Transfection, Construct, Over Expression, Plasmid Preparation, Mutagenesis, Affinity Purification, Binding Assay, Purification

Journal: bioRxiv

Article Title: Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1)

doi: 10.1101/2023.02.09.527921

Figure Lengend Snippet: ( A ) Confocal images of HEK293 cells stably overexpressing MAPPER-GFP (green) transfected with Myc-tagged ESYT1 wild-type and mutant constructs (red) following treatment with DMSO or 1 μM TG to increase intracellular Ca 2+ concentration. Yellow regions within the images represent overlap of MAPPER (green) and Myc-tagged ESYT1 (red), suggesting localization of ESYT1 at ER-PM junctions. The overlap is significantly more extensive following TG treatment of HEK293-MAPPER cells overexpressing wild-type ESYT1 rather than the mutant constructs. ( B-G ) Effect of intracellular Ca 2+ increases on GPR133 surface expression ( B, D, F ) and cAMP levels ( C, E, G ). ( B-C ) TG Treatment of HEK293T cells stably expressing GPR133 transfected with vector, full-length ESYT1 wild-type or the mutant D724A. Bars represent mean ± SEM of 4 to 7 experiments. ( B ) TG treatment had no effect on GPR133 surface expression in GPR133 expressing HEK293T cells transfected with vector, ESYT1 or D724A compared to treatment with DMSO (paired t-test, p>0.05). ( C ) TG treatment significantly increased cAMP levels in GPR133 expressing HEK293T cells transfected with vector and ESYT1 compared to treatment with DMSO (paired t-test, GPR133 + vector: DMSO vs TG, p=0.0210; GPR133 + ESYT1: DMSO vs TG, p=0.0189). TG treatment did not affect GPR133 signaling following transfection of D724A (paired t-test, p>0.05). ns, not significant. ( D-E ) TG Treatment of HEK293T cells transduced with shSCR or shESYT1 to knockdown ESYT1. Bars represent mean ± SEM of 4 experiments. ( D ) TG treatment did not affect GPR133 surface expression compared to treatment with DMSO in GPR133 expressing HEK293T cells transduced with shSCR or shESYT (paired t-test, p>0.05). ( E ) TG treatment significantly increased cAMP concentrations compared to treatment with DMSO in HEK293T cells overexpressing GPR133 and transduced with shSCR (paired t-test, p=0.018). TG treatment had no effect on cAMP levels compared to DMSO following overexpression of GPR133 and knockdown of ESYT1 (paired t-test, p>0.05). ns, not significant. ( F-G ) TG treatment of HEK293T cells stably expressing GPR133 transfected with ESYT1 deletion mutants ΔC2C, ΔC2E or ΔC2C+E. Bars represent mean ± SEM of 4 to 5 experiments. ( F ) Treatment with TG had no effect on GPR133 surface expression in GPR133 expressing HEK293T cells transfected with ΔC2C, ΔC2E or ΔC2C+E compared to treatment with DMSO (paired t-test, p>0.05). ( G ) TG treatment did not affect cAMP concentrations compared to treatment with DMSO in GPR133 expressing HEK293T cells transfected with ΔC2C, ΔC2E or ΔC2C+E (paired t-test, p>0.05). ns, not significant.

Article Snippet: Fusion constructs of wild-type GPR133-BioID2 and mutant GPR133 (H543R/T545A)-BioID2 were created using the MCS-13X Linker-BioID2-HA (addgene #80899) ( Kim et al ., 2016 ) plasmid and subsequently subcloned into the lentiviral backbone pLVX-EF1a-mCherry-N1 (Takara, Cat# 631986).

Techniques: Stable Transfection, Transfection, Mutagenesis, Construct, Concentration Assay, Expressing, Plasmid Preparation, Transduction, Knockdown, Over Expression

Journal: bioRxiv

Article Title: Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1)

doi: 10.1101/2023.02.09.527921

Figure Lengend Snippet: ( A ) Confocal images of HEK293T cells transfected with GPR133 alone (green) or co-transfected with GPR133 and Myc-tagged ESYT1 (red). In the co-transfection condition, the majority of transfected cells express both GPR133 and ESYT1 (orange arrowheads). ( B ) Western blot confirms overexpression of GPR133 and ESYT1 in transfected HEK293T cells. ( C ) Representative PLA images from in HEK293T cells transfected with GPR133 or co-transfected with GPR133 and ESYT1. The red PLA signal (arrow) is only present in cells co-transfected with GPR133 and ESYT1. The signal is weaker in cells treated with 1 μM TG compared to cells treated with DMSO. ( D ) Quantification of PLA positive signals (red dots) over DAPI positive cells overexpressing GPR133 and ESYT1. Bars represent mean ± SEM of 3 experiments. The PLA/DAPI ratio is significantly decreased in TG treated cells (paired t-test, p<0.05). ( E ) Optical sections of GPR133+ESYT1 images from the lower panel in ( C ), detecting a strong PLA signal in DMSO-treated cells (arrow), but a weaker signal in TG-treated cells.

Article Snippet: Fusion constructs of wild-type GPR133-BioID2 and mutant GPR133 (H543R/T545A)-BioID2 were created using the MCS-13X Linker-BioID2-HA (addgene #80899) ( Kim et al ., 2016 ) plasmid and subsequently subcloned into the lentiviral backbone pLVX-EF1a-mCherry-N1 (Takara, Cat# 631986).

Techniques: Transfection, Cotransfection, Western Blot, Over Expression

Journal: bioRxiv

Article Title: Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1)

doi: 10.1101/2023.02.09.527921

Figure Lengend Snippet: ( A, B ) GBML109 was transduced with lentivirus for overexpression of GPR133 and shRNA mediated knockdown of ESYT1. ( A ) Western blot analysis using specific antibodies against ESYT1 (top panel) and GPR133 (bottom panel) confirms expression of ESYT1 in GBML109 transduced with the shSCR control and knockdown of ESYT1 following transduction with shESYT1 in cells overexpressing GPR133 or an empty vector control. ( B ) Intracellular cAMP levels in GPR133-expressing GBML109 cells are significantly increased following knockdown of ESYT1 compared the control (paired t-test, p<0.05). Bars represent mean ± SEM of 5 experiments. ( C ) ESYT1 transcript in a publically available GBM single cell RNA-seq (scRNA-seq) database (Single Single Cell Portal of the Broad Institute). ( Ci ) Identification of cellular populations in GBM specimens using tSNE plots. ( Cii ) ESYT1 is transcribed in tumor cells, as well as macrophages, T cells and oligodendrocytes in the tumor microenvironment. ( D ) Kaplan-Meier survival curves from the TCGA GBM dataset as a function of ESYT1 mRNA levels in bulk RNAseq of surgical specimens. Patients in the upper quartile of ESYT1 mRNA levels experience shorter survival (median 329 days) relative to patients in the lower quartile (median 460 days) (logrank Mantel-Cox test, p=0.0413). ( E , F ) Effects of ESYT1 knockdown by lentiviral transduction of shRNA in GBML154. ( E ) Western blot analysis confirms KD of ESYT1 in GBML154. ( F ) Tumorsphere formation is significantly reduced in GBML154 following KD of ESYT1 compared to the control shSCR (paired t-test, p=0.0306). Bars represent mean ± SEM of 3 experiments. ( G , H ) Tumorsphere formation following the CRISPR/Cas9-mediated KO of ESYT1 in GBML83 and GBML154. ( G ) Reduced ESYT1 expression, detected by Western blot, following transduction with an ESYT1 specific CRISPR/Cas9 construct compared to the Rosa26 control. ( H ) Tumorsphere formation is significantly reduced in GBML83 and GBML154 following KO of ESYT1. Overexpression (OE) of ESYT1 in these cells rescues the effect (GBML83: one-way ANOVA F (2,6) =22.32, p=0.0017; Tukey’s post hoc test: Rosa26 vs. ESYT1 KO, p=0.0023; ESYT1 KO vs. ESYT1 KO + ESYT1 OE, p=0.0036; GBML154: one-way ANOVA F (2,6) =10.30, p=0.0115; Tukey’s post hoc test: Rosa26 vs. ESYT1 KO, p=0.0183; ESYT1 KO vs. ESYT1 KO + ESYT1 OE, p=0.0179). Bars represent mean ± SEM of 3 experiments. ns, not significant.

Article Snippet: Fusion constructs of wild-type GPR133-BioID2 and mutant GPR133 (H543R/T545A)-BioID2 were created using the MCS-13X Linker-BioID2-HA (addgene #80899) ( Kim et al ., 2016 ) plasmid and subsequently subcloned into the lentiviral backbone pLVX-EF1a-mCherry-N1 (Takara, Cat# 631986).

Techniques: Transduction, Over Expression, shRNA, Knockdown, Western Blot, Expressing, Control, Plasmid Preparation, RNA Sequencing, CRISPR, Construct

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of B cell receptor-dependent NF-κB signaling by the tumor suppressor KLHL14

doi: 10.1073/pnas.1921187117

Figure Lengend Snippet: KLHL14 and BCR subunits are in close proximity in ABC DLBCL cells. (A) Confocal images of Tet repressor-expressing TMD8 cells retrovirally transduced with cDNAs encoding BioID2-HA-tagged KLHL14. Cells were treated with 1 μg/mL of doxycycline (DOX) for 24 h and stained as indicated. (B) Confocal images of TMD8 cells stained as indicated. (C, Top) Confocal images of PLAs (red) showing interaction of induced KLHL14 with IgM (C, Left), CD79A (C, Middle), and CD79B (C, Right) in Tet repressor-expressing TMD8 cells retrovirally transduced with cDNAs encoding BioID2-HA-tagged KLHL14 and treated with DOX (1 μg/mL) for 24 h. (Bottom) Normalized PLA score for each PLA pair. (D) Normalized PLA score after shRNA knockdown of the indicated genes (see Materials and Methods for detail). Error bars represent SEM, and data are representative of three independent experiments.

Article Snippet: Anti-IgM (Santa Cruz Biotechnology, catalog no. sc-53347, 1:100), anti-CD79A (Santa Cruz Biotechnology, catalog no. sc-20064, 1:100), anti-CD79B (Santa Cruz Biotechnology, catalog no. sc-53210, 1:100), and anti-BioID2 (Novus Biologicals, catalog no. NBP2-59941, 1:100) were used for each PLA pair.

Techniques: Expressing, Transduction, Staining, shRNA

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of B cell receptor-dependent NF-κB signaling by the tumor suppressor KLHL14

doi: 10.1073/pnas.1921187117

Figure Lengend Snippet: KLHL14 decreases stability of the immature BCR glycoforms in the ER. (A, Left) Western blot analysis of PSA (ER associated, Top)- and RCA (Golgi associated, Bottom)-bound fractions of BCR in Tet repressor-expressing TMD8 cells retrovirally transduced with cDNAs encoding BioID2-HA-tagged KLHL14. Cells were pretreated with DOX (1 μg/mL) for 16 h and treated with CHX (50 μg/mL) for the indicated time points before lysis. Right, quantification of BCR protein levels for PSA (ER associated, Top)- and RCA (Golgi associated, Bottom)-bound fractions. (B, Left) Western blot analysis of PSA (ER associated, Top)- and RCA (Golgi associated, Bottom)-bound fractions of BCR in TMD8-Cas9 cells lentivirally transduced with indicated sgRNAs. Cells were treated with CHX (50 μg/mL) for the indicated time points before lysis. (B, Right) Quantification of BCR protein levels for PSA (ER associated, Top)- and RCA (Golgi associated, Bottom)-bound fractions. Ctrl, nontargeting control. Error bars represent SD of triplicates, and data are representative of three independent experiments.

Article Snippet: Anti-IgM (Santa Cruz Biotechnology, catalog no. sc-53347, 1:100), anti-CD79A (Santa Cruz Biotechnology, catalog no. sc-20064, 1:100), anti-CD79B (Santa Cruz Biotechnology, catalog no. sc-53210, 1:100), and anti-BioID2 (Novus Biologicals, catalog no. NBP2-59941, 1:100) were used for each PLA pair.

Techniques: Western Blot, Expressing, Transduction, Lysis

Journal: bioRxiv

Article Title: Unravelling biological processess and EGFR pathway regulation by the receptor-like protein tyrosine phosphatase PTPRH in non-small cell lung cancer

doi: 10.1101/2024.06.13.598886

Figure Lengend Snippet: H2023 and H23 PTPRH knockout cell lines transduced with BioID2-HA-only, PTPRH WT-BioID2-HA, or mock (control) were validated for expression of the constructs and their subcellular localization using immunoblot and immunofluorescence, respectively. A) Immunoblot against the HA-tag for mock (n=1/cell line), BioID2-only (n=2/cell line), and PTPRH WT-BioID2-HA (n=2/cell line) H2023 and H23 KO cells. Bands matching the correct molecular size for BioID2-HA and PTPRH WT-BioID2-HA are indicated by the arrows. B) Immunofluorescence was performed in all groups using antibodies against the HA-tag which are shown in magenta. DAPI staining is shown in blue. Representative cells showing localization of PTPRH WT-BioID2-HA in the plasma membrane and/or cytosol are indicated by arrows. Images are representative fields using 40x magnification, scale bar = 400 pixels. Abbreviations : ab = antibody; IB= immunoblot.

Article Snippet: The coverslips were then incubated with primary antibodies against BioID2 (1:5000, BioFront #BID2-CP-100) or HA tag (1:800, Cell Signaling #3724) diluted in 1% BSA/ PBS-Tween 0.1% for one hour in a humidified chamber at RT.

Techniques: Knock-Out, Transduction, Control, Expressing, Construct, Western Blot, Immunofluorescence, Staining, Membrane

Journal: bioRxiv

Article Title: Unravelling biological processess and EGFR pathway regulation by the receptor-like protein tyrosine phosphatase PTPRH in non-small cell lung cancer

doi: 10.1101/2024.06.13.598886

Figure Lengend Snippet: H2023 and H23 PTPRH knockout cell lines transduced with BioID2-HA-only, PTPRH WT-BioID2-HA, or mock (control) were stained with antibody against the BioID2 tag subcellular localization of protein is shown in green (immunofluorescence). DAPI staining is shown in blue. Representative cells showing localization of PTPRH WT-BioID2-HA in the plasma membrane and/or cytosol are indicated by arrows. Images are representative fields using 40x magnification, scale bar = 400 pixels. Abbreviations : ab = antibody.

Article Snippet: The coverslips were then incubated with primary antibodies against BioID2 (1:5000, BioFront #BID2-CP-100) or HA tag (1:800, Cell Signaling #3724) diluted in 1% BSA/ PBS-Tween 0.1% for one hour in a humidified chamber at RT.

Techniques: Knock-Out, Transduction, Control, Staining, Immunofluorescence, Membrane

Journal: bioRxiv

Article Title: Unravelling biological processess and EGFR pathway regulation by the receptor-like protein tyrosine phosphatase PTPRH in non-small cell lung cancer

doi: 10.1101/2024.06.13.598886

Figure Lengend Snippet: H2023 and H23 PTPRH KO cell lines transduced with BioID2-HA-only, PTPRH WT-BioID2-HA, or mock (negative control) were serum starved for 24h, followed by stimulation with FBS and biotin for 18h. A) Immunoblot against biotin showing the presence of biotinylated proteins in the BioID2-only and PTPRH WT-BioID2-HA groups, with some bands (arrows) showing up exclusively in the PTPRH-WT-BioID2-HA groups. B) Venn diagram of biotinylated proteins identified in each PTPRH WT-BioID2-HA transduced cell line (n=1/cell line) by mass spectrometry. These proteins present a minimum of 3-fold increase in normalized total precursor intensity compared to their respective BioID2-only transduced cell line. C) Candidate PTPRH interactors (n=48) were screened for the presence of reported phospho sites in the PhosphoSitePlus database. The percentage of proteins containing at least one pY or pS/pT sites are shown. D) STRING analysis of candidate PTPRH interactors (n=58). Abbreviations : IB = immunoblot; pY = phospho-Tyrosine; pS/pT = phospho-Serine/phospho-Threonine.

Article Snippet: The coverslips were then incubated with primary antibodies against BioID2 (1:5000, BioFront #BID2-CP-100) or HA tag (1:800, Cell Signaling #3724) diluted in 1% BSA/ PBS-Tween 0.1% for one hour in a humidified chamber at RT.

Techniques: Transduction, Negative Control, Western Blot, Mass Spectrometry

Journal: Molecular Biology of the Cell

Article Title: An improved smaller biotin ligase for BioID proximity labeling

doi: 10.1091/mbc.E15-12-0844

Figure Lengend Snippet: Promiscuous biotinylation by BioID2. (A) The dimensions of E. coli (left; PDB ID 1BIA) and A. aeolicus (right; PDB ID 2EAY) biotin ligases based on prior structural analyses. The catalytic (yellow), ATP-binding (green), and DNA-binding (red) domains. (B) BioID and BioID2 were fused with LaA and expressed in HEK293 cells. Fusion proteins were detected by specific antibodies against BioID or BioID2, respectively (red). Biotinylated proteins were labeled with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). Scale bar, 10 μm. (C) Proteins biotinylated by BioID-LaA, BioID2-LaA, BioID-only, or BioID2-only were detected with HRP-conjugated streptavidin after SDS–PAGE separation. Expression of either promiscuous ligase leads to biotinylation of endogenous proteins (left). Fusion proteins were detected with anti-myc antibodies (right). (D) BioID-human Sun2 or BioID2-human Sun2 were transiently expressed in NIH3T3 cells. Fusion proteins were detected using an anti-Sun2 antibody incapable of detecting murine Sun2. Scale bar, 10 μm. (E) The NE/ER ratio of the mean intensity of BioID-human Sun2 or BioID2-human Sun2 detected with anti-human Sun2. Values are mean ± SEM. We measured 48 nuclei/condition.

Article Snippet: Owing to the difficulty in detecting the N-terminal myc tag by immunofluorescence (Supplemental Figure S4), we generated a chicken anti-BioID2 using purified GST-BioID2 (1:2000; Aves Labs, Tigard, OR).

Techniques: Binding Assay, Labeling, SDS Page, Expressing

Journal: Molecular Biology of the Cell

Article Title: An improved smaller biotin ligase for BioID proximity labeling

doi: 10.1091/mbc.E15-12-0844

Figure Lengend Snippet: BioID2 requires less biotin than does BioID for promiscuous biotinylation. (A) In vitro biotinylation at variable biotin concentrations was performed using purified BioID (left) and BioID2 (right). Values are mean ±SEM. **** p < 0.0001 and ** p < 0.01 as compared with the 50 μM concentration. Each group consisted of three replicates. (B) Cellular biotinylation at variable biotin concentrations was analyzed using cells stably expressing BioID-LaA (left) and BioID2-LaA (right) in culture media supplemented with 10% fetal bovine serum. Biotinylation was measured after incubation with the indicated concentration of biotin for 16 h. (C) Cellular biotinylation at variable biotin concentration was analyzed using cells stably expressing BioID-LaA (left) and BioID2-LaA (right). Biotin-depleted medium was used to inhibit basal biotinylation. Biotinylation by BioID-LaA or BioID2-LaA was analyzed after incubation with the indicated concentration of biotin for 16 h.

Article Snippet: Owing to the difficulty in detecting the N-terminal myc tag by immunofluorescence (Supplemental Figure S4), we generated a chicken anti-BioID2 using purified GST-BioID2 (1:2000; Aves Labs, Tigard, OR).

Techniques: In Vitro, Purification, Concentration Assay, Stable Transfection, Expressing, Incubation

Journal: Molecular Biology of the Cell

Article Title: An improved smaller biotin ligase for BioID proximity labeling

doi: 10.1091/mbc.E15-12-0844

Figure Lengend Snippet: Application of BioID2 to the human Nup107–Nup160 complex. (A) Expression of Nup43-BioID or Nup43-BioID2 biotinylated endogenous proteins at the NPC. The NPCs were labeled with an anti-Nup153 antibody (red). Biotinylated proteins were detected with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). To observe more clearly the NPCs, confocal images were taken at the surface of the NE. Scale bar, 10 μm. (B) Proteins biotinylated by Nup43-BioID and Nup43-BioID2 were detected with HRP-conjugated streptavidin (top). Fusion proteins were detected with anti-HA antibody (middle). BioID- or BioID2-only controls were detected by an anti-myc antibody (bottom). Asterisk indicates predicted migration of Nup96 and Nup107. (C) Model of the Nup107–Nup160 complex based on the previous literature and resolved structures ( Hoelz et al. , 2011 ; Bui et al. , 2013 ). Candidates identified by both Nup43-BioID (middle) and Nup43-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with blue. Modified from Kim et al. (2014) . (D) The full range of NPC candidates, with those identified by both Nup43-BioID (left) and Nup43-BioID2 (right) labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with patterned blue. Modified from Kim et al. (2014) .

Article Snippet: Owing to the difficulty in detecting the N-terminal myc tag by immunofluorescence (Supplemental Figure S4), we generated a chicken anti-BioID2 using purified GST-BioID2 (1:2000; Aves Labs, Tigard, OR).

Techniques: Expressing, Labeling, Migration, Modification

Journal: Molecular Biology of the Cell

Article Title: An improved smaller biotin ligase for BioID proximity labeling

doi: 10.1091/mbc.E15-12-0844

Figure Lengend Snippet: An extended flexible linker increases the number of candidates detected by Nup43-BioID2. (A) Linear model of Nup43-BioID2 and Nup43-Linker-BioID2 fusion proteins. An extended flexible linker consisting of 13 repeats of GGGGS predicted to provide an ∼25-nm extension was inserted between the Nup43 bait and BioID2 ligase. (B) Expression of Nup43-BioID2 or Nup43-Linker-BioID2 led to biotinylation of endogenous proteins at the NPC. NPCs were labeled using an anti-Nup153 antibody (red). Biotinylated proteins were detected with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). Images were taken at the surface of the NE by confocal microscopy. Scale bar, 10 μm. (C) Proteins biotinylated by Nup43-BioID2 and Nup43-Linker-BioID2 were detected with HRP-conjugated streptavidin (top). Fusion proteins were labeled with anti-HA antibody (bottom). (D) Nup107–Nup160 complex candidates identified by both Nup43-BioID2 (middle) and Nup43-Linker-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with blue. (E) For the entire NPC, Nups identified by both Nup43-BioID2 (left) and Nup43-Linker-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with patterned blue.

Article Snippet: Owing to the difficulty in detecting the N-terminal myc tag by immunofluorescence (Supplemental Figure S4), we generated a chicken anti-BioID2 using purified GST-BioID2 (1:2000; Aves Labs, Tigard, OR).

Techniques: Expressing, Labeling, Confocal Microscopy

Journal: bioRxiv

Article Title: 3D matrix adhesion composition facilitates nuclear force coupling to drive invasive cell migration

doi: 10.1101/2021.05.17.443835

Figure Lengend Snippet: (A) Schematic of BioID2 adhesion constructs used in this study. (B) Immunostaining of MDA-MB-231 cell lines stably expressing BioID2 and BioID2-Paxillin on 2D substrates. Scale bar in expanded view = 20 µm, in magnified view = 5 µm. (C) Immunostaining of MDA-MB-231 cells stably expressing 13x-BioID2 or Talin-13x-BioID2. Scale bar in expanded view = 20 µm, in magnified view = 5 µm. (D & E) Protein-protein interaction network showing all proteins identified as being enriched in BioID2-Paxillin (D) or BioID2-13x-Talin (E) compared with corresponding BioID2 control (Fisher-Exact, P < 0.05 Benjamini-Hochberg corrected) (n=3). Interaction network obtained from STRING 10.5 (Experimentally identified interactions only, confidence level > 0.4). Protein enrichment is shown based on total spectral counts normalised to BioID2 identification in each condition. (F) Protein-protein interaction network showing all proteins identified as being enriched in BioID2-βPIX. Proteins were considered to be positively enriched if 3 or more spectra were identified and they were enriched 2 fold vs a BioID only expressing conditional control. Proteins identified in both 2D and 3D conditions are coloured to represent 3D vs 2D total spectral counts normalised to total βPIX spectra for that given condition. Interaction network obtained from STRING (10.5) based on interaction database and experimental data. Edge weight represents interaction confidence score. (High > 0.95, Medium 0.95-0.9, Low 0.9-0.4) (n = 1).

Article Snippet: MDA-MB-231 cells were transfected with BioID2 using a Nucleofector™ II system (Lonza) according to manufacturer’s instructions (Program X-013, Kit V).

Techniques: Construct, Immunostaining, Stable Transfection, Expressing, Protein Enrichment

Journal: bioRxiv

Article Title: 3D matrix adhesion composition facilitates nuclear force coupling to drive invasive cell migration

doi: 10.1101/2021.05.17.443835

Figure Lengend Snippet: (A, B) Biotinlyation time-course with various BioID2-construct expression. Biotin incubation 50 μM for the times indicated. (C) Expression changes identified in MDA-MB-231 cells cultured in 2D vs 3D by RNA-seq. Scatterplots showing 3D vs 2D gene expression as assessed by RNAseq vs. 3D vs 2D protein enrichment in (C) BioID2-Paxillin (D) Talin1-13x-BioID2 datasets. Red points represent genes with expression changes that are statistically significant (P < 0.05, Benjamini-Hochberg corrected). (E) Table showing genes with statistically significant expression changes 3D vs 2D that were identified in either the BioID2-Paxillin or Talin1-13x-BioID2 datasets. (F) Diagram showing coverage of the ‘consensus’ adhesome from by the BioID2-Paxillin & Talin-13x-BioID2 datasets. Blue dashed boxes enclose the 4 signalling axes described by Horton et al., Left to right: actinin-zyxin, Talin-Vinculin, Paxillin-FAK, ILK-kindlin; top: unconnected ‘consensus’ adhesome proteins. Interaction network obtained from STRING 10.5 (Experimental and Database interactions only, confidence > 0.4). Figure corresponds to .

Article Snippet: MDA-MB-231 cells were transfected with BioID2 using a Nucleofector™ II system (Lonza) according to manufacturer’s instructions (Program X-013, Kit V).

Techniques: Construct, Expressing, Incubation, Cell Culture, RNA Sequencing Assay, Protein Enrichment