venus fluorescent protein  (Addgene inc)

Journal: bioRxiv

Article Title: SARS-CoV-2 membrane protein biogenesis

doi: 10.64898/2026.01.19.700281

Figure Lengend Snippet: (A) To investigate TMD interactions within the SARS-CoV-2 M protein, we employed the BlaTM assay. Productive TMD–TMD interactions reconstitute the β-lactamase enzyme, enabling bacterial growth on selective media. The BlaTM 1.2 system was used to evaluate parallel homo- and heterotypic interactions between TMD1 and TMD3. The BlaTM 2.0 system enables testing of antiparallel heterotypic interactions between TMD2 and TMD1 or TMD3. (B) In the BLaTM assay, the LD 50 of the antibiotic ampicillin indicates the strength of the TMD–TMD interaction. Representative examples of dose– response curves used to calculate LD 50 values. Positive control: GpA TMD homodimer (blue). Negative control: CLS TMD (gray). (C) Same as in (B) but for the BlaTM 2.0 assay. Positive control: TMD homodimer of EmrE (blue). Negative control: a mutant that disrupts this interaction (EmrE mut) (gray). (D) LD 50 values for M TMD–TMD interactions. Values are normalized against the positive controls (dark gray; set to 100%). Data are presented as mean ± SD. Solid dots represent individual experiments ( N = 5–15). Above bars: p values below the significance threshold (< 0.05), calculated using one-sample t-tests against a reference value of 100. (E) M-protein oligomerization was assessed via bimolecular fluorescent complementation (BiFC) assay. The N-terminal and C-terminal ends of the VFP (VN and VC, respectively) were fused to the C-termini of M-protein truncates. The specific residues in each truncate are indicated. (F) BiFC relative fluorescence units (RFU) for M truncates and full-length homo-interactions. All values are normalized to the mean RFU of the full-length M-protein interaction (dark gray; set to 100%). Mean ± SD is shown. Solid dots represent individual experiments ( N = 3–15). Above bars: p values below the significance threshold (<0.05) calculated using one-sample t-tests against a reference value of “100”. (G) Structures of M-protein-dimer constructs from our simulations, from left to right: 50mer, 74mer, 107mer, and full-length. Top representations correspond to the start point of the simulations. Bottom representations show an overlay of three independent replicates’ end-points. (H) Quantification of hydrophobic contacts (HCs) from MD simulations. Results presented as the ratio of the average increase in hydrophobic contacts (ΔHCs) per residue increment (Δres) across the 400-ns simulation (Sim. Av.) and at the initial timepoint (Start) for each simulated dimer. Numerical values are displayed on the heatmap. HCs calculated at the simulation start point are also shown. (I) Same as in (H) for hydrogen bonds (HBs).

Article Snippet: To generate BiFC chimeric plasmids including the Nt or Ct of the Venus Fluorescent Protein (VN, VC respectively; Addgene #27097, #22011, a gift from Chang-Deng H) plasmids were modified to clone the M protein truncates at the Nt of the VFP.

Techniques: Positive Control, Negative Control, Mutagenesis, Bimolecular Fluorescence Complementation Assay, Fluorescence, Construct, Residue

Journal: EMBO Reports

Article Title: Synaptotagmin-11 facilitates assembly of a presynaptic signaling complex in post-Golgi cargo vesicles

doi: 10.1038/s44319-024-00147-0

Figure Lengend Snippet: ( A ) Scheme illustrating the principle of BiFC. Complex formation of Syt11-VN with GB2-VC in the presence of KCTD16 leads to the reconstitution of Venus fluorescence. For validation of the GB2-VC/Syt11-VN BiFC in transfected HEK293T cells, see Fig. . ( B ) Representative confocal images of cultured Kctd16 +/+ and Kctd16 −/− hippocampal neurons (DIV10) transfected with Syt11-VN and GB2-VC. Venus BiFC is observed in axons and dendrites of KCTD16 +/+ neurons. In Kctd16 −/− neurons, low background BiFC in the soma is likely due to Venus self-assembly in the ER. Transfected neurons were identified using mCherry as a volume marker. Scale bar: 10 μm. ( C ) Higher magnification of an axon and dendrite of a mature hippocampal neuron (DIV14) transfected with Syt11-VN, GB2-VC, and mCherry as a volume marker. The BiFC complex (Venus) partly co-localized with endogenous synaptophysin in axons (arrowheads). In dendrites, the BiFC complex localized to dendritic shafts and spine necks but not to spine heads, as identified by PSD95 staining. Scale bar: 5 μm. ( D ) BiFC and endogenous PSD95 fluorescence along BiFC positive spines (normalized to the peak fluorescence). The BiFC signal is high in spine necks and absent from spine heads, contrasting with the distribution of PSD95. Data are presented as mean ± SEM from n = 83 spines (4 independent preparations). ( E ) Time-lapse images and a related kymograph of well-separated fluorescent Syt11-VN/GB2-VC complexes moving anterogradely (white arrowhead) and retrogradely (black arrowhead) in an axon. For quantification of kymographs, see Fig. . ( F ) Representative confocal images of hippocampal neurons transfected with Syt11-VN, GB2-VC, and either NPY-mCherry or Rab5-mCherry. The fluorescent Syt11-VN/GB2-VC complex predominantly co-localizes with NPY-mCherry. MAP2 staining identifies dendrites. Higher magnifications of dendrites are shown at the bottom. Arrowheads indicate examples of co-localization. Scale bar: 10 μm. For quantification of co-localization, see Fig. . .

Article Snippet: In the split Venus constructs Syt11-VN, Syt11ΔC2-VN, Syt1-VN and GB2-VC the N-terminal 1-172 (VN) or C-terminal 155-238 (VC) residues of the yellow fluorescent protein Venus (Nagai et al, ) were cloned in frame at the C-terminus of the respective proteins separated by the linker sequence PRARDPPVAT (Armando et al, ; Dinamarca et al, ).

Techniques: Fluorescence, Biomarker Discovery, Transfection, Cell Culture, Marker, Staining

Journal: EMBO Reports

Article Title: Synaptotagmin-11 facilitates assembly of a presynaptic signaling complex in post-Golgi cargo vesicles

doi: 10.1038/s44319-024-00147-0

Figure Lengend Snippet: ( A ) Representative confocal images of HEK293T cells expressing GB2-VC and Syt11-VN tagged with the C-terminal (VC) and N-terminal (VN) fragments of the fluorescent Venus protein (top row). Reconstitution of Venus fluorescence is observed only in cells expressing KCTD16. In control experiments, replacing Syt11-VN with Syt11ΔC2-VN lacking the C2A and C2B domains (middle row) or Syt1-VN (bottom row) does not reconstitute Venus fluorescence. Transfected cells were identified using mCherry. Scale bar: 10 μm. ( B ) Representative Western blots (left) and corresponding quantifications from n = 5 independent experiments (right) of APs with anti-HA antibodies from cell lysates of transfected HEK293T cells expressing the indicated constructs. AP and input lanes were probed with anti-Syt11 (top), anti-KCTD16 (middle), and anti-HA (bottom) antibodies. The presence of VN- or VC-tags on Syt11 and GB2, respectively, does not significantly alter the amounts of KCTD16 ( p = 0.436) and Syt11 ( p = 0.858) co-purified with GB2. Values are presented as mean ± SEM, ns = not significant, unpaired t-test.

Article Snippet: In the split Venus constructs Syt11-VN, Syt11ΔC2-VN, Syt1-VN and GB2-VC the N-terminal 1-172 (VN) or C-terminal 155-238 (VC) residues of the yellow fluorescent protein Venus (Nagai et al, ) were cloned in frame at the C-terminus of the respective proteins separated by the linker sequence PRARDPPVAT (Armando et al, ; Dinamarca et al, ).

Techniques: Expressing, Fluorescence, Control, Transfection, Western Blot, Construct, Purification

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Schematic representation of CBX2-PRC1. (B) A hypothetical model describing how CBX2-PRC1 is assembled into condensates through phase separation. (C) Representative epi-fluorescence images of condensates of individual CBX2-PRC1 components. Scale bars, 5.0 μm. (D) Condensed fraction of CBX2 and PHC1/2/3 quantified from (C). Error bars denote SD. (E–G) Representative epi-fluorescence images of the scaffold CBX2 and the clients. CBX2, at a fixed concentration of 0.5 μM, was mixed with serial dilutions of the clients RING1B (E), MEL18 (F), and PHC1 (F). Scale bars, 5.0 μm. (H–M) Condensed fraction (H–J) and condensate size (K–M) of the scaffold CBX2 and the clients quantified from (E)–(G). Error bars denote SD.

Article Snippet: For the development of the dual knockin cells, a dTAG-Venus insert was synthesized by Thermo Fisher containing a FKBP12 F36V mutant allowing for targeted degradation experiments as well as a gene for the Venus fluorescence protein.

Techniques: Fluorescence, Concentration Assay

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Schematic representation of the CBX-PRC1 complexes. (B) Condensed fraction of the CBX proteins quantified from . Error bars denote SD. (C) Representative epi-fluorescence images of the CBX-PRC1 components. Panels on left: RING1B was unlabeled and not shown. Panels on right: only the RING1B images are shown. Scale bars, 5.0 μm. (D) Box plot of condensed fraction quantified from (C). (E) A hypothetical model describing how individual CBX-PRC1 complexes are assembled to condensates in vitro . (F) Live-cell epi-fluorescence images showing subnuclear localization of the CBX proteins fused with HaloTag treated with and without Dox. Scale bars, 5.0 μm. (G) Box plot of condensed fraction of the CBX proteins quantified from (F). p value is calculated using Student’s t test (**p < 0.01).

Article Snippet: For the development of the dual knockin cells, a dTAG-Venus insert was synthesized by Thermo Fisher containing a FKBP12 F36V mutant allowing for targeted degradation experiments as well as a gene for the Venus fluorescence protein.

Techniques: Fluorescence, In Vitro

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) A hypothetical model describing how condensate composition regulates the partitioning of CBX2-PRC1 components and nucleosomes and the exchange properties of the scaffold CBX2. Colored hexagons are the CBX2-PRC1 clients (magenta) and nucleosomes (green). (B) Representative epi-fluorescence images of CBX2-PRC1 subunits in the four-component (CBX2, RING1B [R], MEL18 [M], and PHC1 [P]) system. Scale bars, 5.0 μm. (C) Box plot of condensed fraction in the four-component system quantified from (B). p value is calculated using Student’s t test (*p < 0.05; **p < 0.01). (D) FRAP curves of CBX2 in the single-component, two-component, three-component, and four-component systems. Error bars denote SD. (E) Example confocal fluorescence images of CBX2 and nucleosomes (Nuc.) in the two-component, three-component, four-component, and five-component systems. Scale bars, 5.0 μm. (F) Box plot of condensed fraction of CBX2 and nucleosomes quantified from (E). p value is calculated using Student’s t test (**p < 0.01). (G) FRAP curves of YFP-CBX2 in the two-, three-, four-, and five-component systems. Error bars denote SD. (H) Representative live-cell epi-fluorescence images of HT-CBX2 in wild-type (WT), Ring1a −/− /b −/− , and Bmi1 −/− /Mel18 −/− mESC lines. Scale bars, 5.0 μm. (I and J) Box plots of condensed fraction (I) and size (J) of HT-CBX2 condensates quantified from (H). p value is calculated using Student’s t test (**p < 0.01). Error bars denote SD. (K) Example confocal images of FRAP of HT-CBX2 in wild-type (WT), Ring1a −/− /b −/− , and Bmi1 −/− /Mel18 −/− mESC lines. Red arrows show condensates to be bleached. Scale bar, 5.0 μm. (L) FRAP curves of HT-CBX2 within and outside condensates in wild-type (WT), Ring1a −/− /b −/− , and Bmi1 −/− /Mel18 −/− mESC lines. Error bars denote SD.

Article Snippet: For the development of the dual knockin cells, a dTAG-Venus insert was synthesized by Thermo Fisher containing a FKBP12 F36V mutant allowing for targeted degradation experiments as well as a gene for the Venus fluorescence protein.

Techniques: Fluorescence

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) A hypothetical model describing how the CBX-PRC1 complexes are assembled into condensates. (B) Representative epi-fluorescence images of condensates of CBX2 and other CBX proteins. CBX2-PRC1 was mixed with CBX4-, CBX6-, CBX7-, and CBX8-PRC1, respectively. Scale bars, 5.0 μm. (C) Box plot of condensed fraction of CBX2 and other CBX proteins quantified from (B). p value is calculated using Student’s t test (*p < 0.05; **p < 0.01). (D) Live-cell epi-fluorescence images of HT-CBX2 and YFP-CBX4/6/7/8 in HeLa cells. Scale bars, 5.0 μm. (E) Condensate-based Pearson correlation coefficient (PCC) of other CBX condensates with CBX2 condensates quantified from (D).

Article Snippet: For the development of the dual knockin cells, a dTAG-Venus insert was synthesized by Thermo Fisher containing a FKBP12 F36V mutant allowing for targeted degradation experiments as well as a gene for the Venus fluorescence protein.

Techniques: Fluorescence

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Schematic representation for CRISPR-Cas9-mediated homologous recombination to insert LoxP sites flanking exon 5 of both alleles of the Cbx2 locus in mESCs. The two scissors indicate the two single-guide RNA (sgRNA)-targeted locations. The red arrows indicate the primers used to verify the insertion and depletion of exon 5. (B) Representative epi-fluorescence images of Cbx2 fl/fl -HT mESCs after administering 4-hydroxytamoxifen (OHT) for different periods of time. Scale bars, 5.0 μm. (C) Box plot of fluorescence (FL) intensity per cell of CBX2-HT quantified from (B). p value is calculated using Student’s t test (**p < 0.01). (D) Schematic representation of dual-knockin mESCs containing Cbx2 fl/fl -HT and Cbx7-Venus-dTAG (left) or Cbx2 fl/fl -HT and dTAG-Venus-Phc1 (right). (E) Live-cell epi-fluorescence images of colocalization of CBX2-HaloTag with CBX7-Venus or Venus-PHC1 in Cbx2 fl/fl -HT/Cbx7-Venus-dTAG or Cbx2 fl/fl -HT/dTAG-Venus-Phc1 mESCs. Scale bars, 5.0 μm. (F) Box plot of condensed fraction of CBX2-HT, CBX7-Venus, and Venus-PHC1 quantified from (E). p value is calculated using Student’s t test (**p < 0.01). (G) Box plot of condensate-based Pearson correlation coefficient (PCC) of CBX2-HT versus CBX7-Venus or Venus-PHC1 quantified from (E). (H) Live-cell epi-fluorescence images of CBX7-Venus and Venus-PHC1 in dual-knockin mESCs treated with or without OHT. Scale bars, 5.0 μm. (I) Box plot of condensed fraction of CBX7-Venus and Venus-PHC1 quantified from (H). p value is calculated using Student’s t test (**p < 0.01). (J) Box plot of fluorescence intensity per cell of CBX7-Venus and Venus-PHC1 quantified from (H). p value is calculated using Student’s t test.

Article Snippet: For the development of the dual knockin cells, a dTAG-Venus insert was synthesized by Thermo Fisher containing a FKBP12 F36V mutant allowing for targeted degradation experiments as well as a gene for the Venus fluorescence protein.

Techniques: CRISPR, Homologous Recombination, Fluorescence, Knock-In