yfp variant venus  (Addgene inc)

Journal: Current biology : CB

Article Title: Protein interaction analysis provides a map of the spatial and temporal organization of the ciliary gating zone

doi: 10.1016/j.cub.2017.06.044

Figure Lengend Snippet: (A) Schematic of BiFC assay. The N- and C-terminal halves of a YFP (half-stars labeled N or C), when fused to proteins of interest, can reconstitute a fluorescent protein only when brought in close proximity via the interactions of their fusion partners. TZ, transition zone proteins. MT, axonemal microtubules. (B) Localization of Nup62-YC (left) and KIF17-YN (right) expressed in the absence of a BiFC partner. Arl13b-Cer or acetylated α-tubulin (AcTubulin) was used as a cilium marker. Images are of cropped regions containing the cilium, contrast-enhanced for viewing, with a schematic of the observed fluorescence localization shown below each fluorescence image. (C–E) Representative images and schematic depictions show the locations of BiFC interactions detected for Nup62-YC with (C) kinesin-2 motor KIF17-YN, (D) KIF17 with mutated CLS (KIF17ΔCLS-YN), or (E) non-ciliary protein (YN-SAH-FKBP). Nup62-YC was detected with an antibody to the HA tag (Nup62-YC-HA) whereas the KIF17 and SAH constructs were detected with an antibody to the Myc tag. See Table S1 for full description of constructs. See Figure S1 for uncropped images. (F) Quantification of the locations of BiFC interactions. The number of cells observed for each BiFC location category is indicated on the bar graph. (G–I) Time course of interaction between Nup62 and KIF17. Nup62-FRB-Cer and mCit-KIF17-FKBP were co-expressed and the cells were fixed after treatment with (G) ethanol (- Rapamycin control), (H) 10 min Rapamycin, or (I) 30 min Rapamycin. Graphs show the mean fluorescence of Nup62-FRB-Cer at the base or tip of the cilium (normalized to the fluorescence at the base) or the mean fluorescence of mCit-KIF17-FKBP at the base or tip of the cilium (normalized to the fluorescence at the tip). *p<0.01 compared to control (-rapamycin) by Student’s t-test. Error bars, S.D. n = 10–14 cells each.

Article Snippet: We began our studies using fragments of the YFP variant Venus [N-terminal aa1–172, (Addgene plasmid #22010) and C-terminal aa155–238/A206K (Addgene plasmid #22011), gifts from Dr. Chang-Deng Hu] because of its high efficiency in BiFC [ 59 ].

Techniques: Bimolecular Fluorescence Complementation Assay, Labeling, Marker, Fluorescence, Construct, Control

Journal: Current biology : CB

Article Title: Protein interaction analysis provides a map of the spatial and temporal organization of the ciliary gating zone

doi: 10.1016/j.cub.2017.06.044

Figure Lengend Snippet: (A) Schematic of BiFC assay. The N- and C-terminal halves of a YFP (half-stars labeled N or C), when fused to proteins of interest, can reconstitute a fluorescent protein only when brought in close proximity via the interactions of their fusion partners. TZ, transition zone proteins. MT, axonemal microtubules. (B) Localization of Nup62-YC (left) and KIF17-YN (right) expressed in the absence of a BiFC partner. Arl13b-Cer or acetylated α-tubulin (AcTubulin) was used as a cilium marker. Images are of cropped regions containing the cilium, contrast-enhanced for viewing, with a schematic of the observed fluorescence localization shown below each fluorescence image. (C–E) Representative images and schematic depictions show the locations of BiFC interactions detected for Nup62-YC with (C) kinesin-2 motor KIF17-YN, (D) KIF17 with mutated CLS (KIF17ΔCLS-YN), or (E) non-ciliary protein (YN-SAH-FKBP). Nup62-YC was detected with an antibody to the HA tag (Nup62-YC-HA) whereas the KIF17 and SAH constructs were detected with an antibody to the Myc tag. See Table S1 for full description of constructs. See Figure S1 for uncropped images. (F) Quantification of the locations of BiFC interactions. The number of cells observed for each BiFC location category is indicated on the bar graph. (G–I) Time course of interaction between Nup62 and KIF17. Nup62-FRB-Cer and mCit-KIF17-FKBP were co-expressed and the cells were fixed after treatment with (G) ethanol (- Rapamycin control), (H) 10 min Rapamycin, or (I) 30 min Rapamycin. Graphs show the mean fluorescence of Nup62-FRB-Cer at the base or tip of the cilium (normalized to the fluorescence at the base) or the mean fluorescence of mCit-KIF17-FKBP at the base or tip of the cilium (normalized to the fluorescence at the tip). *p<0.01 compared to control (-rapamycin) by Student’s t-test. Error bars, S.D. n = 10–14 cells each.

Article Snippet: BiFC constructs We began our studies using fragments of the YFP variant Venus [N-terminal aa1–172, (Addgene plasmid #22010) and C-terminal aa155–238/A206K (Addgene plasmid #22011), gifts from Dr. Chang-Deng Hu] because of its high efficiency in BiFC [ 59 ].

Techniques: Bimolecular Fluorescence Complementation Assay, Labeling, Marker, Fluorescence, Construct, Control