ha-fv2e encoding expression vector pc4m) argent regulated homodimerization kit  (ARIAD Inc)

Journal: eLife

Article Title: Celsr1 adhesive interactions mediate the asymmetric organization of planar polarity complexes

doi: 10.7554/eLife.62097

Figure Lengend Snippet: ( A ) Illustration of FKBP-dimer system. Protein of interest is tagged with FKBP domain. Cell-permeable agent B/B homodimerizer (dimerizer) forces FKBP domains together. ( B ) Cultured keratinocytes were transfected with Celsr1 Crsh -FKBP-HA, switched to 1.5 mM calcium for 24 hr, and treated with ethanol (control) or dimerizer for 2 hr prior to the end of the calcium switch to induce dimerization of the FKBP domain fused to Celsr1 Crsh . Yellow arrows highlight position of the cell–cell interface between Celsr1 Crsh -FKBP-HA expressing cells. ( C ) Border enrichment quantification for Celsr1 Crsh -FKBP-HA. Ethanol, n = 14; Dimerizer, n = 12. One-tailed, unpaired t -test, **p=0.0057. ( D ) Cell mixing assay where cells were transfected separately with either Celsr1 WT -GFP or Celsr1 Crsh -FKBP-HA and mixed. Shown are representative examples of mixed-cell junctions between Celsr1 WT -GFP (top cell in pair, green) and Celsr1 Crsh -FKBP-HA (bottom cell in pair, red) treated with ethanol (control) or dimerizer. Scale bars, 10 µm. Figure 6—source data 1. Data accompanying .

Article Snippet: Celsr1 Crsh -FKBP-HA was generated by replacing EGFP in Celsr1 Crsh -GFP ( ) with HA-tagged FKBP (pC 4 -Fv 1 E; ARGENT Regulated Homodimerization Kit, Ariad) digested with HindIII and NotI sites.

Techniques: Cell Culture, Transfection, Control, Expressing, One-tailed Test

Journal: eLife

Article Title: Celsr1 adhesive interactions mediate the asymmetric organization of planar polarity complexes

doi: 10.7554/eLife.62097

Figure Lengend Snippet: Additional representative examples of cell borders of neighboring cells both expressing transfected constructs from cis -dimerization FBKP experiment. Images cropped and rotated to display border with horizontal orientation. Mouse keratinocytes were transfected with constructs indicated, switched to 1.5 mM calcium for 24 hr along with treatment of either ethanol (control) or dimerizer (B/B homo-dimerizer) for the final 2 hr of the calcium switch to induce FKBP domain dimerization. ( A ) Transfection of just Celsr1 Crsh -FKBP-HA. ( B ) Co-transfection of both Celsr1 WT -GFP with Celsr1 Crsh -FKBP-HA. ( C ) Mixed culture of cells transfected with either Celsr1 WT -GFP or Celsr1 Crsh -FKBP-HA. Scale bar, 10 µm.

Article Snippet: Celsr1 Crsh -FKBP-HA was generated by replacing EGFP in Celsr1 Crsh -GFP ( ) with HA-tagged FKBP (pC 4 -Fv 1 E; ARGENT Regulated Homodimerization Kit, Ariad) digested with HindIII and NotI sites.

Techniques: Expressing, Transfection, Construct, Control, Cotransfection

Journal: Traffic (Copenhagen, Denmark)

Article Title: The Translocation Selectivity of the Kinesins that Mediate Neuronal Organelle Transport

doi: 10.1111/j.1600-0854.2011.01325.x

Figure Lengend Snippet: (A) Addition of an Fv domain to the C-termini of KIF13A or 13B enabled induction of homodimerization by addition of AP20187. (B) Addition of AP20187 to a cell expressing KIF13B393Fv caused the kinesin to accumulate at neurite tips. Just before drug addition (0 min), GFP-KIF13B393Fv was distributed diffusely throughout the cell. Within 5 min of adding AP20187 (10 nM), GFP-KIF13B393Fv began to accumulate at neurite tips. The contrast was inverted so that fluorescent signals appear dark on a light background. (C) Distribution of GFP-KIF13A392Fv, GFP-KIF13B393Fv, and GFP-KIF13B393T311M_Fv after incubation with 10nM AP20187. KIF13A392Fv accumulated in axonal but not dendritic tips whereas KIF13B393Fv accumulated in both axonal and dendritic tips. KIF13B393Fv, with a mutation that disrupts microtubule-stimulated ATPase activity, failed to accumulate at either axonal or dendritic tips. Fluorescent signal from the kinesins is shown in red and soluble mRFP in green. Dashed arcs indicate dendritic tips and asterisks indicate axonal tips. Scale bars= 20μm. (D) Translocation preferences of KIF13A392Fv and KIF13B393Fv constructs, based on the analysis of 18 or 15 cells quantified as in Fig. 2. Data points for KIF1A393 and KIF5C559 were taken from the experiment shown in Figure 2I. The image of GFP-KIF13B393T311M_Fv was assembled from 4 microscopic fields as described in Materials and Methods.

Article Snippet: For the KIF13 dimerization assay, an ARGENT TM Regulated Homodimerization Kit (Fv) was obtained from ARIAD Pharmaceuticals (Cambridge, MA).

Techniques: Expressing, Incubation, Mutagenesis, Activity Assay, Translocation Assay, Construct