Journal: Development (Cambridge, England)

Article Title: The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network

doi: 10.1242/dev.185827

Figure Lengend Snippet: Bmp and canonical Wnt signalling do not directly synergise to induce proximal identity in the optic vesicle. (A) 3D surface reconstructions of the chick optic vesicle/cup from stages HH10-HH16. The horizontal plane of sectioning is indicated for stage HH10. (B,C) An activator-inhibitor type Turing network. (B) A slow-diffusing activator morphogen drives its own production and that of a faster-diffusing inhibitor morphogen, which inhibits the activator. (C) The network yields a molar excess of activator over inhibitor at their common source, but an excess of inhibitor away from their source. (D) Schematic representation of a horizontal section through the stage HH10 chick optic vesicle identifying neighbouring tissues, anterior-posterior axis and proximal-distal axis. OV, optic vesicle; PLE, presumptive lens ectoderm; POM, periocular mesenchyme; FB, forebrain; MB, midbrain. (E-K) The HH10 optic vesicle is polarised along a proximal-distal axis. Horizontal sections reveal polarised expression of the marker genes (E) Pax6 , (F) Vsx2 , (G) Wnt2b , (H) Mitf , (I) Tgfb2 and (J) Fst . (K) Bmp4 is expressed in the overlying presumptive lens ectoderm. (L-O) RT-QPCR analysis of proximal and distal marker gene expression following 16 h exposure to (L) Bmp4 only, (M) Bmp4 and BIO (a canonical Wnt agonist) in combination, (N) BIO only and (O) DMSO carrier control. Values plotted are Log10(mean fold change)±s.e.m. Red guidelines indicate the levels of ±2-fold change in gene expression. * P <0.05, ** P <0.01 (Student's paired t -test). Scale bars: 150 µm.

Article Snippet: After blocking overnight at 4°C in PBTS (BSA, Triton X-100 and goat serum), explants were incubated in mouse anti-Pax6 primary antibody (diluted 1:50 in PBTS; Developmental Studies Hybridoma Bank #PAX6) for 3 days then washed in PBS-Tween.

Techniques: Expressing, Marker, Quantitative RT-PCR, Gene Expression, Control

Journal: Development (Cambridge, England)

Article Title: The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network

doi: 10.1242/dev.185827

Figure Lengend Snippet: Bmp signalling is required for Pax6 gene expression in the distal optic vesicle. (A) DNA expression constructs were injected into the lumen of the anterior neural tube of stage HH8 chick embryos and electroporated to transfect a single prospective optic vesicle, the other serving as an untransfected internal control. Embryos were cultured for 10-12 h overnight until stage HH10 when they were analysed. (B) Schematic showing the domain structure of the major Pax6 isoform compared with the truncated dominant-negative Pax6 (dnPax6). PAI and RED, DNA-binding subdomains that make up the N-terminal paired domain; HD, DNA-binding homeodomain; P/S/T, C-terminal proline/serine/threonine-rich transactivation domain. Antisense RNA probes against C- or N-terminal sequences respectively detect endogenous Pax6 transcripts only or endogenous Pax6 and dnPax6 together. (C) The sectional area of Pax6 gene expression was measured and compared between electroporated and non-electroporated optic vesicles for each embryo. Log10(fold change) was plotted for embryos electroporated with GFP control construct, Smad6+GFP construct or dnPax6+GFP construct. Red guidelines indicate the level of ±2-fold change in sectional expression area. * P <0.05; n.s. indicates P ≥0.05 (one-way ANOVA with Tukey's post-hoc test). (D-G) Endogenous Pax6 gene expression following transfection with (D) GFP control, (E) Smad6+GFP and (F,G) dnPax6+GFP, and (D′-G′) anti-GFP immunofluorescence showing the location of (D′) GFP transfected cells, (E′) Smad6+GFP transfected cells and (F′,G′) dnPax6+GFP transfected cells. Scale bars: 100 µm. Immunofluorescence in G′ is heavily quenched by strong in situ staining. Optic vesicles are indicated by broken outlines.

Article Snippet: After blocking overnight at 4°C in PBTS (BSA, Triton X-100 and goat serum), explants were incubated in mouse anti-Pax6 primary antibody (diluted 1:50 in PBTS; Developmental Studies Hybridoma Bank #PAX6) for 3 days then washed in PBS-Tween.

Techniques: Gene Expression, Expressing, Construct, Injection, Control, Cell Culture, Dominant Negative Mutation, Binding Assay, Transfection, Immunofluorescence, In Situ, Staining

Journal: Development (Cambridge, England)

Article Title: The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network

doi: 10.1242/dev.185827

Figure Lengend Snippet: Pax6 function is required for expression of Tgfb2 and Fst . (A-C′) Tgfb2 gene expression was assessed 12 h after electroporation of GFP or dnPax6+GFP into a single optic vesicle. (A) Sectional areas of Tgfb2 gene expression were measured and compared between electroporated and non-electroporated optic vesicles for each embryo. Log10(fold change) was plotted for each embryo. Red guidelines indicate the level of ±2-fold change in sectional expression area. (B,C) Tgfb2 gene expression following electroporation with (B) GFP control and (C) dnPax6+GFP, and (B′,C′) anti-GFP immunofluorescence showing the location of (B′) GFP transfected cells and (C′) dnPax6+GFP transfected cells. (D-F) Fst expression was assessed 12 h after electroporation with GFP or dnPax6+GFP. (D) Sectional areas of Fst gene expression were measured and compared between electroporated and non-electroporated optic vesicles for each embryo. Log10(fold change) was plotted for each embryo. Red guidelines indicate the level of ±2-fold change in sectional expression area. (E,F) Fst gene expression following electroporation with (E) GFP control and (F) dnPax6+GFP, and (E′,F′) anti-GFP immunostaining showing the location of (E′) GFP transfected cells and (F′) dnPax6+GFP transfected cells. Optic vesicles are indicated by broken outlines. * P <0.05 (Welch's two-sample t -test). Scale bars: 100 µm.

Article Snippet: After blocking overnight at 4°C in PBTS (BSA, Triton X-100 and goat serum), explants were incubated in mouse anti-Pax6 primary antibody (diluted 1:50 in PBTS; Developmental Studies Hybridoma Bank #PAX6) for 3 days then washed in PBS-Tween.

Techniques: Expressing, Gene Expression, Electroporation, Control, Immunofluorescence, Transfection, Immunostaining

Journal: Development (Cambridge, England)

Article Title: The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network

doi: 10.1242/dev.185827

Figure Lengend Snippet: Reaction-diffusion modelling of the Pax6 / Fst / Tgfb2 gene network. (A) Summary of Model A, in which Pax6 drives expression of both Fst and Tgfb2 , whereas Fst inhibits Tgfβ2 function via sequestration. Slow diffusion of Fst was postulated to result in local inhibition of Tgfβ2 at the source of Pax6 / Tgfb2 / Fst expression. Conversely, fast diffusion of Tgfβ2 was postulated to drive lateral activation of its downstream signalling pathway away from the Pax6 / Fst / Tgfb2 -expressing region. (B,B′) 1D numerical simulation of Model A, in which Pax6 expression is regionally restricted throughout. For all simulations, units of space, time and molecular concentrations are arbitrary. The vertical y -axis represents the anterior-posterior axis of the hemispherical optic vesicle, which is divided into anterior-proximal, distal and posterior-proximal domains. The plots depict the time evolution ( x -axis) for 1D spatial distributions ( y -axis) of (B) Pax6 and (B′) the activated Tgfβ2:Tgfβ-receptor signalling complex. (C) Summary of Model B in which Fst:Tgfβ2 complex quickly diffuses and dissociates, while Tgfβ2 additionally inhibits Pax6 transcriptional activator function. (D,D′) 1D numerical simulation of Model B in which Pax6 expression is initially homogenous but noisy. The plots depict spontaneous generation of (D) a Pax6+ ‘distal pole’ flanked by (D′) Tgfβ2:Tgfβ-receptor+ ‘proximal poles’. (E,E′) 1D numerical simulation of Model B with a larger tissue size resulting in (E) multiple Pax6+ ‘distal poles’ interspersed with (E′) Tgfβ2:Tgfβ-receptor+ ‘proximal poles’. (F) Confocal section of an HH10 tg(membrane-GFP) embryo showing optic vesicle size prior to explant culture. (G) Confocal section of a fixed optic vesicle explant showing the collapsed tissue following 16 h culture. Cell nuclei are stained with DAPI. (H,H′) 2D numerical simulation of Model B within an explant-shaped domain (Model C). (H) The initial distal-high to proximal-low Pax6 pattern is (H′) dynamically re-polarised along the longest axis of the explant. (I,I′) Partially dissected optic vesicle in which the distal end was fluorescently labelled with (I) DiO, corresponding to (I′) the Pax6+ pole revealed by immunofluorescent staining. (J,J′) Explant experiment in which (J) the distal pole of the optic vesicle was labelled with DiO during dissection. (J′) Following overnight culture, Pax6 expression has re-polarised relative to the former proximal-distal axis. Scale bars: 50 µm in F,G,J; 100 µm in I.

Article Snippet: After blocking overnight at 4°C in PBTS (BSA, Triton X-100 and goat serum), explants were incubated in mouse anti-Pax6 primary antibody (diluted 1:50 in PBTS; Developmental Studies Hybridoma Bank #PAX6) for 3 days then washed in PBS-Tween.

Techniques: Diffusion-based Assay, Expressing, Inhibition, Activation Assay, Membrane, Staining, Dissection

Journal: Development (Cambridge, England)

Article Title: The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network

doi: 10.1242/dev.185827

Figure Lengend Snippet: Shh positional information and Tgf β -mediated self-organisation position the Pax6+ pole in cultured explants. (A,A′) The Pax6+ pole re-aligns with the dorsal-ventral axis in explanted optic vesicles. Maximal projections of (A) Pax6 immunofluorescence normalised to DAPI and (A′) ventrally targeted GFP in a whole-mount explant. (B) Summary of Model D in which a ventral-high to dorsal-low Shh gradient inhibits Pax6 expression. The pharmacological compounds used in functional experiments are also indicated (broken lines). (C,C′) 2D numerical simulation of Model D showing (C) the ventral-high Shh gradient (C′) Pax6 re-polarisation. (D,D′) 2D numerical simulation of Model D showing (D) reversal of the Shh gradient and (D′) corresponding reversal of Pax6 polarity. (E,E′) 2D numerical simulation of Model D with Tgfβ loss of function (Tgfb LOF) showing (E) the ventral high Shh gradient and (E′) the resulting Pax6 distribution. (F,F′) Optic vesicle explants were cultured with 10 μM SIS3 for 16 h. (F) Maximum projection of Pax6 immunofluorescence normalised to DAPI. (F′) 1D profile plot of Pax6 abundance along the longest (horizontal) axis of the explant. (G,G′) 2D numerical simulation of Model D with Shh loss of function (Shh LOF) showing (G) absence of Shh positional information and (G′) the resulting Pax6 distribution. (H,H′) Optic vesicle explants were cultured with 2.5 μM cyclopamine for 16 h. (H) Maximum projection of Pax6 immunofluorescence normalised to DAPI. (H′) 1D profile plot of Pax6 abundance along the longest (horizontal) axis of the explant. (I,I′) 2D numerical simulation of Model D with both Tgfβ loss of function and Shh loss of function showing (I) absence of Shh positional information and (I′) the resulting Pax6 distribution. (J,J′) Optic vesicle explants were cultured with both 10 μM SIS3 and 2.5 μM cyclopamine for 16 h. (J) Maximum projection of Pax6 immunofluorescence normalised to DAPI. (J′) 1D profile plot of Pax6 abundance along the longest (horizontal) axis of the explant. Scale bars: 50 μm.

Article Snippet: After blocking overnight at 4°C in PBTS (BSA, Triton X-100 and goat serum), explants were incubated in mouse anti-Pax6 primary antibody (diluted 1:50 in PBTS; Developmental Studies Hybridoma Bank #PAX6) for 3 days then washed in PBS-Tween.

Techniques: Cell Culture, Immunofluorescence, Expressing, Functional Assay

Journal: Development (Cambridge, England)

Article Title: The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network

doi: 10.1242/dev.185827

Figure Lengend Snippet: Fst gene function is required for correct optic vesicle polarisation via distal inhibition of Tgfβ signalling. (A) Schematic showing domain structures encoded by naturally occurring Fst transcripts. The shorter Fst 300 is generated by alternative splicing. SP, 28 amino acid signal peptide cleaved co-translationally; NTD, N-terminal domain; FSD, follistatin domain; AT, acidic tail. (B) Western blot validation of Fst 315 and Fst 300 protein knockdown by FstMO but not by StdMO in cultured chick embryo cells. (C-G′) Sectional area of Pax6 gene expression was assessed 12 h after co-electroporation of single optic vesicles with control/experimental morpholinos plus various gene expression constructs. (C) Sectional area of Pax6 gene expression was measured and compared between electroporated and non-electroporated optic vesicles for each embryo. Log10(fold change) was plotted for each embryo. Red guidelines indicate the level of ±2-fold change in sectional expression area. (D-G) Pax6 gene expression following co-electroporation of (D) standard control morpholino (StdMO)+GFP, (E) Fst morpholino (FstMO)+GFP, (F) FstMO+Fst gene expression construct and (G) FstMO+Smad7 gene expression construct. (D′-G′) FITC-labelled (D′) StdMO fluorescence, (E′) FstMO fluorescence, (F′) FstMO fluorescence and (G′), FstMO fluorescence, showing the location of transfected cells. (H-J′) Sectional area of Vsx2 gene expression was assessed 12 h after co-electroporation of single optic vesicles with control/experimental morpholino. (H) Sectional area of Vsx2 gene expression was measured and compared between electroporated and non-electroporated optic vesicles for each embryo. Log10(fold change) was plotted for each embryo. Red guidelines indicate the level of ±2-fold change in sectional expression area. (I,J) Vsx2 gene expression following co-electroporation of (I) StdMO+GFP and (J) FstMO+GFP, and (I′,J′) FITC-labelled (I′) StdMO fluorescence and (J′) FstMO fluorescence showing the location of transfected cells. Optic vesicles are indicated by broken outlines. * P <0.05; ** P <0.01 (C, one-way ANOVA with Tukey's post-hoc test; H, Welch's two-sample t -test). Scale bars: 100 µm.

Article Snippet: After blocking overnight at 4°C in PBTS (BSA, Triton X-100 and goat serum), explants were incubated in mouse anti-Pax6 primary antibody (diluted 1:50 in PBTS; Developmental Studies Hybridoma Bank #PAX6) for 3 days then washed in PBS-Tween.

Techniques: Inhibition, Generated, Alternative Splicing, Western Blot, Biomarker Discovery, Knockdown, Cell Culture, Gene Expression, Electroporation, Control, Construct, Expressing, Fluorescence, Transfection

Journal: Development (Cambridge, England)

Article Title: The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network

doi: 10.1242/dev.185827

Figure Lengend Snippet: Proposed Pax6 / Fst / Tgfb2 network function during optic vesicle polarisation in vivo . (A) At the prospective distal pole, Pax6 expression is promoted by upstream Bmp and reinforced via auto-regulation. Pax6 drives distal expression of Fst , Tgfb2 and downstream Vsx2 . A molar excess of slow-diffusing Fst over Tgfβ receptors is postulated to reversibly sequester Tgfβ2 into fast-diffusing Fst:Tgfβ2 complexes. (B) At the prospective proximal vesicle, dissociation of fast-diffusing Fst:Tgfβ2 complexes is postulated to release Tgfβ2. A molar excess of Tgfβ receptors over slow-diffusing Fst then permits receptor activation by Tgfβ2, causing functional inhibition of Pax6 and induction of proximal markers Wnt2b and Mitf . Interactions indicated by broken lines may be indirect.

Article Snippet: After blocking overnight at 4°C in PBTS (BSA, Triton X-100 and goat serum), explants were incubated in mouse anti-Pax6 primary antibody (diluted 1:50 in PBTS; Developmental Studies Hybridoma Bank #PAX6) for 3 days then washed in PBS-Tween.

Techniques: In Vivo, Expressing, Activation Assay, Functional Assay, Inhibition

Journal: Nature Communications

Article Title: Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes

doi: 10.1038/s41467-023-38923-6

Figure Lengend Snippet: a Split kinesin recruitment assay used to identify MBNL1-kinesin associations. Cargoes are recruited in a retrograde or anterograde direction in a rapalog-dependent manner, depending on choice of motor complex. b Representative images showing recruitment of EGFP-MBNL1 (green) to the centrosome by Kif1bα, but not Kif1bβ, using Myc-tagged, FRB-kinesin tail fusions (blue) co-expressed with BicD2-FKBP or KIF1A motor-FKBP (red) in N2A cells. Scale bar = 10 µm. c A modified kinesin recruitment assay that uses BicD2-kinesin tail fusions to further characterize MBNL1-kinesin associations. d Representative images and quantitation in N2A cells of EGFP-MBNL1 (green) at the centrosome when co-expressed with BicD2-kinesin tail fusions (red); nuclei (blue) labeled with DAPI; Kif1bα: n = 93 cells (50, 20, and 23 cells in each replicate), Kif1bβ: n = 81 cells (25, 16, and 40 cells in each replicate), Kif1c: n = 112 cells (53, 19, and 40 cells in each replicate); across 3 biological replicates. Scale bar = 10 µm. Bars represent median, box outlines represent upper and lower quartiles, whiskers represent 10th and 90th percentiles. Dotted line represents no enrichment. **** p < 0.0001 by two-tailed Mann–Whitney U test. e Representative dual color time lapse images of EGFP-MBNL1 (green) and mScarlet-I-kinesins (red) in live 8 DIV primary mouse cortical neurons. Arrows denote co-transported, anterograde particles of MBNL with Kif1bα (left) and Kif1c (right), whereas Kif1bβ particles lack MBNL (center); time (sec) indicated in top or bottom right. Scale bar = 5 µm. Kymographs are displayed below still frames obtained from movies. White arrows denote co-transport events. Anterograde transport is shown from left to right. Scale bar = 10 µm. f Fraction of mScarlet-I-kinesin granules showing co-transport with EGFP-MBNL1; across 4 biological replicates. Bars represent mean. * p < 0.05, ** p < 0.01 by one-way ANOVA with Tukey’s post-hoc test. g Western blots against RFP, MBNL1, and GAPDH following co-immunoprecipitation of mScarlet-Kif fusions with quantitation normalized to input MBNL1-EGFP protein; across 3 biological replicates. Lysates were collected after transient expression in N2A cells. Bars denote mean. ** p < 0.01, ***<0.001 by one-way ANOVA with Tukey’s post-hoc test. Source data provided as a .

Article Snippet: Membranes were blocked in 5% BSA in PBS before being incubated overnight at 4 °C with rabbit ɑ-MBNL1 antibody (1:1000, Millipore Sigma), mouse ɑ-MBNL1 antibody (1:100, DHSB, 4A8), mouse ɑ-RFP antibody (1:1000, Proteintech, 5F8), rabbit ɑ-Kif1c (1:500, Proteintech, 12760-1-AP), or mouse ɑ-KIF1bβ (1:500, EMD Millipore, MABC309).

Techniques: Modification, Quantitation Assay, Labeling, Two Tailed Test, MANN-WHITNEY, Western Blot, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes

doi: 10.1038/s41467-023-38923-6

Figure Lengend Snippet: a Experimental design used to deplete MBNLs or over-express kinesin tail dominant negatives in differentiated N2A cells, followed by fractionation of soma and neurites. b Bars showing change in localization of mRNAs from neurite to soma in N2A expressing CTG480 as compared to CTG0, as a function of CLIP tag density per kilobase of 5′ and 3′ UTR. * p < 0.01, ** p < 0.005 by two-tailed rank-sum test. Data represented as median values ± SEM. Data comes from one biological replicate. c Venn diagram showing overlap of mRNA targets mis-localized from neurite to soma upon perturbation by CTG480 relative to CTG0 or Kif1bα/Kif1c tails relative to Kif1bβ tail. P values computed by two-tailed Fisher’s Exact test, and enrichment of overlap was computed assuming independence between conditions. d The ratio of nucleolin mRNA between neurite and soma across conditions, as assessed by RNAseq. e Distances of Ncl and Gapdh HCR FISH spots from the soma of each cell across conditions. Distances were normalized as a fraction of total neurite length, and the 95th percentile of distances within each cell is plotted as an individual gray circle. Each cell (Ncl - CTG0: n = 8 cells, CTG480: n = 8 cells, Kif1bβ: n = 9 cells, Kif1bα: n = 9 cells, Kif1c: n = 10 cells; Gapdh - CTG0: n = 8 cells, CTG480: n = 9 cells, Kif1bβ: n = 9 cells, Kif1bα: n = 8 cells, Kif1c: n = 8 cells; across 3 biological replicates) contained >150 Ncl spots and >300 Gapdh spots. Bars represent the median across cells. * p < 0.05, ** p < 0.01 by two-tailed Mann–Whitney U test. f Representative HCR FISH images for Ncl and Gapdh (red) with CTG repeats or dominant negative kinesin tails (green) in differentiated N2A cells. Scale bar = 10 µm. Source data provided as a .

Article Snippet: Membranes were blocked in 5% BSA in PBS before being incubated overnight at 4 °C with rabbit ɑ-MBNL1 antibody (1:1000, Millipore Sigma), mouse ɑ-MBNL1 antibody (1:100, DHSB, 4A8), mouse ɑ-RFP antibody (1:1000, Proteintech, 5F8), rabbit ɑ-Kif1c (1:500, Proteintech, 12760-1-AP), or mouse ɑ-KIF1bβ (1:500, EMD Millipore, MABC309).

Techniques: Fractionation, Expressing, Two Tailed Test, MANN-WHITNEY, Dominant Negative Mutation

Journal: Nature Communications

Article Title: Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes

doi: 10.1038/s41467-023-38923-6

Figure Lengend Snippet: a Schematic of EGFP-MBNL1 mutants tested. b Representative images of centrosome enrichment for each mutant (green) as tested with BicD2-Kif1c tail fusions (red) in N2A cells. Nuclei labeled with DAPI (blue). c Quantitation of centrosome enrichment for each mutant. MBNL1-41 : n = 112 cells (53, 19, and 40 cells in each replicate), MBNL1-41-RIM : n = 146 cells, MBNL1-Δ3, ΔC RIM : n = 106 cells, MBNL1-41-CM : n = 182 cells. d Quantitation of centrosome enrichment for EGFP-ZnF1/2 RIM and EGFP-ZnF3/4 RIM mutants using BicD2 kinesin tail fusions. MBNL1-ZnF1,2 RIM - Kif1bα: n = 39 cells, Kif1bβ: n = 29 cells, Kif1c: n = 40 cells; MBNL1-ZnF3,4 RIM - Kif1bα: n = 42 cells, Kif1bβ: n = 39 cells, Kif1c: n = 44 cells. e Representative images of centrosome enrichment for Tis11d (green); f Rbfox2 (green); g Cpsf4 (green); and ( h ) ZC3H14 (green) with BicD2 kinesin tail fusions (red) in N2A cells. i Centrosome enrichment quantitation for Tis11d - Kif1bα: n = 29 cells, Kif1bβ: n = 49 cells, Kif1c: n = 49 cells; and ( j ), Rbfox2 - Kif1bα: n = 36 cells, Kif1bβ: n = 35 cells, Kif1c: n = 36 cells; and ( k ), Cpsf4 - Kif1bα: n = 95 cells, Kif1bβ: n = 88 cells, Kif1c: n = 81 cells; and ( l ), ZC3H14 - Kif1bα: n = 43 cells, Kif1bβ: n = 40 cells, Kif1c: n = 25 cells. Bars represent median, box outline represent upper and lower quartiles, whiskers represent 10th and 90th percentiles. Dotted line represents no enrichment (=1). Scale bars = 5 µm. **** p < 0.0001, ** p < 0.01, * p < 0.05 by two-tailed Mann–Whitney U test. Nuclei labeled with DAPI (blue). All experiments performed across 3 biological replicates. Source data provided as a .

Article Snippet: Membranes were blocked in 5% BSA in PBS before being incubated overnight at 4 °C with rabbit ɑ-MBNL1 antibody (1:1000, Millipore Sigma), mouse ɑ-MBNL1 antibody (1:100, DHSB, 4A8), mouse ɑ-RFP antibody (1:1000, Proteintech, 5F8), rabbit ɑ-Kif1c (1:500, Proteintech, 12760-1-AP), or mouse ɑ-KIF1bβ (1:500, EMD Millipore, MABC309).

Techniques: Mutagenesis, Labeling, Quantitation Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes

doi: 10.1038/s41467-023-38923-6

Figure Lengend Snippet: a MS2 reporter system used to image single RNA molecules and reconstitute activities of MBNL domains to localize RNAs by fusion to MCP-Halo. Fusions generated include full-length MBNL1 or truncated MBNL1 without C-terminal tail and/or exon 3; ZnFs in these contexts were all RIM mutants incapable of binding RNA. b Particle tracks (multi-colored) from C2C12 myoblasts stably expressing MCP-Halo-RIM, MCP-Halo-ΔC RIM, or MCP-Halo-Δ3, ΔC RIM. c Inferred ensemble log diffusion coefficients (µm 2 /s) and maximum distance (µm) traveled for particles in each condition; MCP-Halo-RIM: 1233 particle tracks, MCP-Halo-ΔC RIM: 1039 particle tracks, MCP-Halo-Δ3, ΔC RIM: 934 particle tracks. Median per cell log diffusion coefficients (µm 2 /s) and maximum distance travelled for each particle (µm); n = 11 cells per condition across 3 biological replicates. d Schematic and particle tracks (multi-colored) from movies of C2C12 myoblasts stably expressing MCP-Halo or MCP-Halo-Cterm. e Inferred ensemble log diffusion coefficients (µm 2 /s) and maximum distance (µm) traveled for particles in each condition; MCP-Halo: 1205 particle tracks, MCP-Halo-Cterm: 885 particle tracks. Median per cell log diffusion coefficients (µm 2 /s) and maximum distance (µm); n = 16 cells per condition across 3 biological replicates. f Enrichment of MS2 reporter mRNA as assessed by RT-qPCR from membrane and cytosolic fractions of C2C12 myoblasts in the presence of MCP-Halo or MCP-Halo-Cterm, n = 5 biological replicates for each condition. g Schematic of MS2 reporter system used to reconstitute kinesin-dependent transport of RNA via MBNL1 zinc fingers. h Representative images from C2C12 myoblasts of HCR FISH (red) against the MS2 mRNA reporter in the presence of BicD2-Kif1c (green) and Halo-MCP fusions. Nuclei labeled with DAPI (blue). i Quantitation of the fraction of SunTag-MS2 RNA molecules present at the centrosome relative to the whole cell across each condition. Scale bars = 5 µm. * p < 0.05, ** p < 0.01, *** p < 0.001 by two-tailed Mann–Whitney U test. Source data provided as a .

Article Snippet: Membranes were blocked in 5% BSA in PBS before being incubated overnight at 4 °C with rabbit ɑ-MBNL1 antibody (1:1000, Millipore Sigma), mouse ɑ-MBNL1 antibody (1:100, DHSB, 4A8), mouse ɑ-RFP antibody (1:1000, Proteintech, 5F8), rabbit ɑ-Kif1c (1:500, Proteintech, 12760-1-AP), or mouse ɑ-KIF1bβ (1:500, EMD Millipore, MABC309).

Techniques: Generated, Binding Assay, Stable Transfection, Expressing, Diffusion-based Assay, Quantitative RT-PCR, Membrane, Zinc-Fingers, Labeling, Quantitation Assay, Two Tailed Test, MANN-WHITNEY

Journal: Oncology Letters

Article Title: Role of specific DNA mutations in the peripheral blood of colorectal cancer patients for the assessment of tumor stage and residual disease following tumor resection

doi: 10.3892/ol.2016.5078

Figure Lengend Snippet: List of KRAS and BRAF mutations covered by the KRAS/BRAF mutation analysis kit (EntroGen).

Article Snippet: Determination of KRAS and BRAF mutation status was performed using the KRAS/BRAF Mutation Analysis kit (EntroGen, Tarzana, CA, USA) for a quantitative polymerase chain reaction (qPCR) assay based on allele-specific PCR.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

doi: 10.1371/journal.pone.0253687

Figure Lengend Snippet: Overview of the EGFR phenotypes detected by the SensiScreen ® EGFR Liquid assay (PentaBase ApS) versus the ctEGFR Mutation Detection Kit (EntroGen) using cohort II and agreement or disagreement between platforms (N = 34).

Article Snippet: The main targeted techniques are [ , ]: Quantitative real-time PCR (RT-PCR) (e.g. Therascreen ® (QIAGEN) [ ], cobas ® (Roche) [ , ]), ctEGFR Mutation Detection Kit (EntroGen)) [ ], droplet digital PCR (ddPCR) [ , , ], beads emulsion amplification and magnetics quantitative PCR (BEAMing qPCR) [ ] and ultra-deep NGS [ ] (e.g. the Ion Torrent ® based OncomineTM Lung cfDNA Assay (Thermo Fisher Scientific)).

Techniques: Mutagenesis