beta-catenin (armadillo) antibody  (Developmental Studies Hybridoma Bank)

Journal: eLife

Article Title: Differential regulation of the proteome and phosphoproteome along the dorso-ventral axis of the early Drosophila embryo

doi: 10.7554/eLife.99263

Figure Lengend Snippet: ( A ) Top: color-coded schematic of the cell populations along the dorso-ventral axis of a Drosophila embryo during gastrulation: blue, 'dorsal': ectoderm and amnioserosa; magenta, 'lateral': neuroectoderm; green, mesectoderm; yellow 'ventral': mesoderm. Middle: examples of dorso-ventral fate determinants of the domains shown in the top panel. Bottom: dorso-ventral and anterior-posterior axes for reference in panel B. ( B ) RNA in situ hybridisations using probes for genes expressed in dorsal ( dpp ), lateral ( sog ) and ventral ( snail ) cell populations in wild type ( w 1118 ) and embryos derived from mothers mutant for dorso-ventral patterning genes ( gd 9 , dorsalized, Tl rm9 / Tlrm rm10 lateralized, Toll 10B / def and spn27A ex / def both ventralized). Notice the expansion of dpp, sog and snail expression in dorsalized, lateralized and ventralized embryos respectively. Arrow indicates remaining neuroectodermal polarity in ventralized embryos. ( C ) Images (confocal, max-projected) of physical cross-sections from heat-fixed embryos stained using antibodies against β-Catenin/Armadillo (green) and Snail (magenta). D is dorsal domain; L is lateral domain; V is ventral domain. Scale bar is 50 μm. ( D ) log2 intensity (top) and log2 fold change (FC, bottom) of proteins in wild type, dorsalized (blue), lateralized (magenta), and ventralized (yellow) embryos. Bars depict mean and standard error of the mean across replicates. Absence of a dot indicates the protein was not detected or log2FC calculation not feasible; absence of error bars in log2 intensity indicates protein was detected only in a single biological replicate. Dotted line indicates log2FC = 0. Mean log2 intensity values were compared using one-way ANOVA, followed by pairwise unpaired t-test comparisons (FDR corrected). Significance: * is p<0.05, ** is p<0.01, *** is p<0.001. See for ANOVA and pairwise comparison p-values. ( E ) Cross-section images (two-photon, single sections) showing Myosin Light Chain (sqh-mCherry) distribution in living wild type, dorsalized, lateralized and ventralized embryos. Insets show magnified ectopic sqh-mCherry signal distribution in wild type vs. ventralized embryos. Scale bar is 50 μm for full view and 25 μm for insets. ( F ) Images (spinning disk, max-projected) showing myosin distribution in the sub-apical domain of living wild type, dorsalized, lateralized and ventralized embryos along their dorso-ventral axis. Scale bar is 25 μm.

Article Snippet: Primary antibodies used were: mouse anti α-tubulin 1:1000 (T6199, clone 6-11B-1, Sigma), mouse anti acetylated-α-tubulin FITC conjugated 1:250 (sc23950, Santa Cruz Biotechnology), rat anti tyrosinated-α-tubulin 1:250 (MAB1864-I, clone YL1/2, EMD Millipore/Merck), mouse anti-Armadillo/β-Catenin 1:50 (N27A1, Developmental Studies Hybridoma Bank), rabbit anti Snail ( ) 1:500.

Techniques: In Situ, Derivative Assay, Mutagenesis, Expressing, Staining, Comparison

Journal: eLife

Article Title: Differential regulation of the proteome and phosphoproteome along the dorso-ventral axis of the early Drosophila embryo

doi: 10.7554/eLife.99263

Figure Lengend Snippet: ( A ) Images (confocal, max-projected) of physical cross-sections of heat-fixed embryos showing the transition from stage 5 (late cellularization) to stage 6, and the progression of gastrulation during stage 6 (ventral furrow formation), stained using antibodies against β-Catenin/Armadillo (green in top panel; greyscale in bottom panel) and Snail (magenta in top panel). ( B ) Scheme explaining the strategy for proteomic and phosphoproteomic experiments. Synchronized collections of embryos from wild type or mutant mothers, representing the dorsal ectodermal (dorsalized: gd 9 ), neuroectodermal (lateralized: Tl rm9 / Tlrm rm10 ) and mesodermal (ventralized: Toll 10B / def & spn27A ex / def ) cell populations were allowed to develop for 2 hr 30' at 25 °C, manually selected under visual inspection for 15' to secure the collection of stage 6 embryos and immediately frozen in liquid nitrogen. For the phosphoproteomic experiments, we used SILAC metabolic labeling to optimize the quantification strategy. ( C ) Workflow for quantitative label free proteomics using high-pH offline peptide fractionation followed by LC-MS/MS of individual fractions. ( D ) Workflow for quantitative SILAC-phosphoproteomic analyses. Phosphopeptide enrichment was performed using TiO2 beads, and LC-MS/MS based identification and quantification. For the mass-spectrometry analysis of each replicate, we combined equal amounts of protein from wild type embryos labeled with SILAC-Lys 13/6, and embryos of the target genotype (500 μg target genotype: 500 μg SILAC wild type). ( E ) Histogram showing the distribution of the number of protein groups with respect to the log2 fold change between the intensity values measured for a given protein group in SILAC wild type vs. non-SILAC wild type embryos. H stands for heavy (or SILAC) and L stands for light (or non-SILAC) in pilot runs.

Article Snippet: Primary antibodies used were: mouse anti α-tubulin 1:1000 (T6199, clone 6-11B-1, Sigma), mouse anti acetylated-α-tubulin FITC conjugated 1:250 (sc23950, Santa Cruz Biotechnology), rat anti tyrosinated-α-tubulin 1:250 (MAB1864-I, clone YL1/2, EMD Millipore/Merck), mouse anti-Armadillo/β-Catenin 1:50 (N27A1, Developmental Studies Hybridoma Bank), rabbit anti Snail ( ) 1:500.

Techniques: Staining, Mutagenesis, Multiplex sample analysis, Labeling, Peptide Fractionation, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics, Mass Spectrometry