dd solubilizer  (TaKaRa)

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet:

Article Snippet: D/D solubilizer (DDS) , Clontech , 635034.

Techniques: Recombinant, CRISPR, Transfection, Plasmid Preparation, Electron Microscopy, Reverse Transcription, Software

Journal: Cells

Article Title: BARS Influences Neuronal Development by Regulation of Post-Golgi Trafficking

doi: 10.3390/cells11081320

Figure Lengend Snippet: BARS regulates trafficking of TfR. ( a – h ) A series of confocal images showing the distribution of ectopically-expressed TfR-GFP-FM4 alone or after co-transfection with BARS D355A. The distribution of TfR-GFP-FM4 is indicated at different time points after the addition of DD solubilizer. Inset, distribution of the Golgi apparatus marker TGN38 (red). In control cells (GFP-transfected) most of the TfR-GFP labeling disappeared from the Golgi region 300 min after the addition of DD ( d ); the arrowheads in ( d ) indicate the presence of the labeling close to the dendritic plasma membrane. By contrast, in neurons co-expressing the BARS fission-defective mutant most of the labeling remains in the Golgi area ( h ). Scale bar: 10 µm. ( i ) Graphs showing the fluorescent intensity (FI) ratio between TfR-GFP labeling in dendrites and the Golgi area at 15, 60, 180, and 300 min after the addition of DD solubilizer in control and in neurons expressing BARS D355A mutant. ( j – o ) A series of confocal images showing the distribution of ectopically-expressed TfR-GFP-FM4 after co-transfection with BARS S147A (blue in k) or BARS S147A G172E (blue in n). The distribution of TfR-GFP-FM4 is indicated at different time points after the addition of DD solubilizer. Inset, distribution of the Golgi apparatus marker TGN38 (red). Scale bar: 10 µm. ( p – r ) A series of confocal images showing the distribution of ectopically-expressed TfR-GFP-FM4 after co-transfection with BARS WT (blue in ( q )). The distribution of TfR-GFP-FM4 is indicated at different time points after the addition of DD solubilizer. Inset, distribution of the Golgi apparatus marker TGN38 (red). Scale bar: 10 µm. ( s ) Graphs showing the fluorescent intensity (FI) ratio between TfR-GFP labeling in dendrites and the Golgi area at 15 and 60 min after the addition of DD solubilizer in control and in neurons co-expressing BARS WT or different mutants. For all experiments 9 to 15 neurons were quantified pooled from at least three independent cultures. Graphs represent mean ± S.E.M.; * p < 0.05, ** p < 0.01; one-way ANOVA and Tukey’s post hoc test.

Article Snippet: The FM domains are variants of FKBP (FK506-binding protein), which are able to reversibly self-aggregate into homodimers that can disaggregate within minutes after addition of the membrane permeable drug DD solubilizer (Takara Bio Inc., Kusatsu City, Japan, Cat Number: 635053).

Techniques: Cotransfection, Marker, Control, Transfection, Labeling, Clinical Proteomics, Membrane, Expressing, Mutagenesis

Journal: Cells

Article Title: BARS Influences Neuronal Development by Regulation of Post-Golgi Trafficking

doi: 10.3390/cells11081320

Figure Lengend Snippet: BARS regulates trafficking of ApoER2. ( a – f ) A series of confocal images showing the distribution of ectopically-expressed FM4-ApoER2-GFP alone or after co-transfection with BARS S174A and visualized 0, 15 and 60 min after the addition of DD solubilizer. Inset, distribution of the Golgi apparatus marker TGN38. Scale bar: 10 µm. ( g ) Graph showing the fluorescent intensity (FI) ratio between ApoER2-GFP labeling in dendrites and the Golgi area 15 and 60 min after the addition of DD solubilizer in control- and BARS S147A-expressing neurons. For all experiments, 9 neurons were quantified pooled from at least three independent cultures. Graphs represent mean ± S.E.M.; *** p < 0.001, Student’s t -test. ( h ) A confocal image showing the distribution of ApoER2 in a cortical neuron of mouse brain (embryonic day E18.5) transfected by IUE with sc-shRNA BARS-HcRED (scScramble,) plus ApoER2-GFP; note that the green labeling localized to a discrete area in the cell body and within the apical oriented neurite (image rotated 90º clockwise with respect to the ventral zone–cortical plate axis). ( i ) Idem as in ( h ), but from a mouse brain (embryonic day E18.5) electroporated with sh-RNA-BARS-HcRED (shBARS). ( h’ , i’ ) High magnification views of the inserts shown in ( h , i ). Scale bar: 5 µm. ( j ) A confocal image showing the distribution of ApoER2-GFP in a cell from a brain co-electroporated with RFP. ( k ) Idem as in ( j ), but from a brain electroporated with ApoER2 plus BARS S147A G172E. Scale bar: 10 µm. ( j’ , k’ ) High magnification views of the inserts shown in ( j , k ). Scale bar: 5 µm. ( l ) Graph showing the fluorescent intensity (FI) ratio between ApoER2-GFP labeling in the soma and the apical oriented neurite in cortical neurons after IUE of shScramble or shBARS. ( m ) Graph showing the fluorescent intensity (FI) ratio between ApoER2-GFP labeling in the soma and the apical oriented neurite in cortical neurons after IUE of RFP or RFP plus BARS S147A G172E. For all experiments 13 to 20 neurons were analyzed from 3 independent IUE for each experimental condition. Graphs represent mean ± S.E.M.; * p < 0.05, ** p < 0.01, Student’s t -test.

Article Snippet: The FM domains are variants of FKBP (FK506-binding protein), which are able to reversibly self-aggregate into homodimers that can disaggregate within minutes after addition of the membrane permeable drug DD solubilizer (Takara Bio Inc., Kusatsu City, Japan, Cat Number: 635053).

Techniques: Cotransfection, Marker, Labeling, Control, Expressing, Transfection, shRNA