psbtet bp ultraid arf6  (Addgene inc)

Journal: bioRxiv

Article Title: The combination of optogenetic-induced protein aggregation and proximity biotinylation assays strongly implicates endolysosomal proteins in the early stages of α-synuclein aggregation

doi: 10.1101/2024.10.16.618762

Figure Lengend Snippet: ( A ) Schematic representation of the UltraID-LIPA constructs, composed of Cry2olig (green) fused to mCherry (red) at the C-terminal region. Wild-type α-syn (yellow) or TDP-43 (orange) were then fused at the N-terminal region of Cry2olig. Finally, UltraID was tagged to the N-terminal region of either α-syn (UltraID-LIPA-α-syn), TDP-43 (UltraID-LIPA-TDP-43) or directly to Cry2olig (UltraID-LIPA-Empty). ( B ) Schematic representation of the UltraID proximity biotinylating labelling. UltraID-fused constructs, expressed as monomers, undergo a rapid conversion to oligomers upon exposure to the blue light. After adding exogenous biotin, UltraID catalyses the biotinylating the proteins in contact or in close proximity (10 nm) with the bait protein. Most of the proteins will be transient or permanent interactors of the bait protein and are different depending on the status of the protein (either monomers or oligomers). ( C ) Schematic representation of the general workflow, from the cells to the LC/MS proteomic screening. The specific proteome of the newly formed α-syn oligomers is determined using several conditions: UltraID-LIPA-α-syn monomers (not exposed to blue light) are directly compared toUltraID-LIPA-α-syn oligomers formed under the blue light control. Other LIPA oligomeric constructs were used to control for the specificity of the α-syn interactome, namely UltraID-LIPA-Empty and UltraID-LIPA-TDP-43. The cells are then lysed, and the proteins are extracted using a RIPA lysis method combined with sonication. Extracted material was then digested, and the small peptides tagged with the biotin were captured using Streptavidin-Sepharose beads. The collected peptides were finally purified and analyzed using LC/MS.

Article Snippet: Resulting lentiviral vectors were transformed using One Shot™ Stbl3™ Chemically Competent cells (Thermofisher, Cat. C737303) and amplified using GenElute™ HP Plasmid Maxiprep Kit (Sigma, Cat. NA0310-1KT). pDEST Flp-In UltraID-3xFLAG-5’ was generated by amplifying the UltraID-3xFLAG insert from pSF3 UltraID vector (Addgene, Cat. 172878) using mutagenic primers to induce NheI (before the insert; primer: 5’-ATA TGC TAG CAT GTT CAA GAA CCT GAT CTG GCT G-3’) and NsiI (after the insert; primer: 5’-ATA TAT GCA TCT TCT CCT TGA ACT TCT TCA GG-3’) cleavage sites.

Techniques: Construct, Liquid Chromatography with Mass Spectroscopy, Control, Lysis, Sonication, Purification

Journal: bioRxiv

Article Title: The combination of optogenetic-induced protein aggregation and proximity biotinylation assays strongly implicates endolysosomal proteins in the early stages of α-synuclein aggregation

doi: 10.1101/2024.10.16.618762

Figure Lengend Snippet: ( A ) Confocal maximum intensity projections images of Flp-In T-REx HEK293T cells stably expressing UltraID-LIPA-α-syn construct. Cells were exposed to blue light for 1h, 3h and 12h to induce the aggregation. Scale bar = 10 µm. ( B ) Box plots graphs showing a quantification comparing the cells stably expressing the UltraID-tagged aggregates and the non-tagged, evaluating the number of cells bearing aggregates after several timepoints of illumination. Statistical differences were assessed with a two-Way ANOVA test, comparing the non-tagged cells and the tagged-cells overtime (ns = not significant). ( D ) Confocal maximum intensity projections images of HEk cells overexpressing LIPA-α-syn or UltraID-LIPA-α-syn, exposed (12h) or no to the blue light and stained for the pathological α-syn phosphorylation at the residue S129 (pS129). In both conditions we were able to detect pS129 staining after 12h of blue light exposure. Scale bar = 10 µm. ( D ) Representative Western blot membrane from Flp-In T-REx HEK293T cells stably expressing UltraID constructs, probed for Streptavidin. Cells were cultured with Biolock in the culture medium to block any endogenous biotinylation. For the biotinylation assay, biolock was removed and exogenous biotin was added at 50 µM to the cells whenever they were also exposed to blue light, for 30 min. Note that Streptavidin shows positive staining only in the conditions where exogenous biotin was added for all the UltraID constructs, with a signal accumulated at the expected size of the constructs (∼90/125/110 kDA, for UltraID-LIPA-Empty, UltraID-LIPA-α-syn and UltraID-LIPA-TDP-43, respectively). (

Article Snippet: Resulting lentiviral vectors were transformed using One Shot™ Stbl3™ Chemically Competent cells (Thermofisher, Cat. C737303) and amplified using GenElute™ HP Plasmid Maxiprep Kit (Sigma, Cat. NA0310-1KT). pDEST Flp-In UltraID-3xFLAG-5’ was generated by amplifying the UltraID-3xFLAG insert from pSF3 UltraID vector (Addgene, Cat. 172878) using mutagenic primers to induce NheI (before the insert; primer: 5’-ATA TGC TAG CAT GTT CAA GAA CCT GAT CTG GCT G-3’) and NsiI (after the insert; primer: 5’-ATA TAT GCA TCT TCT CCT TGA ACT TCT TCA GG-3’) cleavage sites.

Techniques: Stable Transfection, Expressing, Construct, Staining, Residue, Western Blot, Membrane, Cell Culture, Blocking Assay, Cell Surface Biotinylation Assay

Journal: bioRxiv

Article Title: The combination of optogenetic-induced protein aggregation and proximity biotinylation assays strongly implicates endolysosomal proteins in the early stages of α-synuclein aggregation

doi: 10.1101/2024.10.16.618762

Figure Lengend Snippet: Confocal maximum intensity projections images of Flp-In T-REx HEK293T cells stably expressing UltraID-LIPA-α-syn construct. Cells were exposed to blue light for 1h, 3h and 12h to induce the aggregation. Scale bar = 10 µm.

Article Snippet: Resulting lentiviral vectors were transformed using One Shot™ Stbl3™ Chemically Competent cells (Thermofisher, Cat. C737303) and amplified using GenElute™ HP Plasmid Maxiprep Kit (Sigma, Cat. NA0310-1KT). pDEST Flp-In UltraID-3xFLAG-5’ was generated by amplifying the UltraID-3xFLAG insert from pSF3 UltraID vector (Addgene, Cat. 172878) using mutagenic primers to induce NheI (before the insert; primer: 5’-ATA TGC TAG CAT GTT CAA GAA CCT GAT CTG GCT G-3’) and NsiI (after the insert; primer: 5’-ATA TAT GCA TCT TCT CCT TGA ACT TCT TCA GG-3’) cleavage sites.

Techniques: Stable Transfection, Expressing, Construct

Journal: Communications Biology

Article Title: Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

doi: 10.1038/s42003-022-03604-5

Figure Lengend Snippet: Blots of lysates of HeLa cells transiently transfected with the indicated constructs and incubated with 50 μM biotin for 10 min. Biotinylation was analyzed using IRDye680-labeled streptavidin and expression levels of the fusion proteins with antibodies against the Myc tag as indicated. a Comparison microID, ultraID-4 and -5, and TurboID. b Comparison microID, microID L41P-only, microID R40G/L41P (=ultraID), microID S24C/R40G/L41P (=ultraID-4 with K169R reverted to WT) and TurboID.

Article Snippet: Plasmids for bacterial and mammalian expression of microID and ultraID are available at Addgene (Plasmid IDs 172878–172881).

Techniques: Transfection, Construct, Incubation, Labeling, Expressing, Comparison

Journal: Communications Biology

Article Title: Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

doi: 10.1038/s42003-022-03604-5

Figure Lengend Snippet: a Blots of lysates of S. cerevisiae strains expressing the indicated Asc1 fusion proteins and incubated or not (−) with 10 μM biotin for the indicated times. The fusion proteins were expressed from plasmids in a Δ asc1 strain, and the ASC1 wild-type strain carrying the empty vector served as a control. Biotinylation was analyzed using HRP-labeled streptavidin and expression levels of the fusion proteins with antibodies against Asc1 as indicated. b Blots of lysates of E. coli cells transformed with expression plasmids for ultraID, microID, TurboID, or the control plasmid. The cells had been incubated (+) or not (−) with biotin for 16 h. The arrowhead indicated the expected size for bacterial GAPDH, and the asterisk (*) indicates a non-specific cross-reactivity that was used as a loading control.

Article Snippet: Plasmids for bacterial and mammalian expression of microID and ultraID are available at Addgene (Plasmid IDs 172878–172881).

Techniques: Expressing, Incubation, Plasmid Preparation, Control, Labeling, Transformation Assay

Journal: Communications Biology

Article Title: Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

doi: 10.1038/s42003-022-03604-5

Figure Lengend Snippet: a Experimental set-up for the side-by-side comparison of overnight labeling with BioID vs. 10 min labeling with ultraID or TurboID. b Venn diagram displaying the proteomic dataset sizes and overlap of the significant hits from a , the gene names of the dataset overlap are specified. c Relative abundance, assessed with iBAQ values, of endogenous biotinylated carboxylases and main interacting partners of AGO2 (TNRC6A & B) and RAB11A (RAB11FIP1 & 5), obtained from HeLa cells expressing TurboID-Ago2 and ultraID-Ago2 (left) or TurboID-Rab11and ultraID-Rab11 (right) after streptavidin pulldowns with no biotin addition. Bars are mean values, n = 3 (Ago2 cell lines) or 4 (Rab11 cell lines).

Article Snippet: Plasmids for bacterial and mammalian expression of microID and ultraID are available at Addgene (Plasmid IDs 172878–172881).

Techniques: Comparison, Labeling, Expressing

Journal: Communications Biology

Article Title: Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

doi: 10.1038/s42003-022-03604-5

Figure Lengend Snippet: a Blots of lysates of P19 cells stably expressing ultraID-Ago2, of P19 cells knock-out for endogenous γ1-COP and rescued by γ1-COP-ultraID, and of P19 cells knock-out for γ2-COP and rescued by γ2-COP-ultraID lines. The fusion proteins were detected with antibodies against γ1- and γ2-COP and biotinylation with IRDye680-labeled streptavidin. b Experimental set-up for the determination of the membrane-associated interactome of γ1-COP and γ2-COP. c Volcano plots of the proteins identified by LC-MS/MS with the mock vs. BFA samples. Significant hits ( p value <0.01 and log 2 fold change >2 in this analysis, and p value <0.01 and log 2 fold change >2 in the γ-COP vs. Ago2 comparison under mock conditions), are indicated with gene names in red. d Proteins identified as membrane interactors of γ1-COP and γ2-COP (see text).

Article Snippet: Plasmids for bacterial and mammalian expression of microID and ultraID are available at Addgene (Plasmid IDs 172878–172881).

Techniques: Stable Transfection, Expressing, Knock-Out, Labeling, Membrane, Liquid Chromatography with Mass Spectroscopy, Comparison