Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A,B) Wnt-responsive luciferase TOPFLASH assay measuring TCF/LEF reporter activity in (A) MCF7 and (B) RKO cells following Wnt3A transfection and siPP1β and siMYPT3 transfection. Data are presented as mean ± SEM with letters above representing significant difference from corresponding column, (P<0.01). (C) Western blot analysis of total cellular levels of E-cad, β-cat, MYPT3, Wnt3A and PP1β. β-tub was used as a loading control. (D) Western blot analysis of β-cat in cytoplasmic (Cyto), membranous (Mem), chromatin-bound (CB), and whole cell extract (WCE) fractions in MCF7 cells. GAPDH, Na + /K + ATPase, and Histone 3 were used as loading controls for corresponding fractions. Complete fraction, see . (E) Quantification of relative levels of β-cat from each fraction taken from (D), normalized to Control + siScramble conditions. Data shown as mean ± SEM (n = 4 biological replicates), ns = not significant, *P<0.05.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, and probed against the following primary antibodies: rabbit anti-β-catenin (1:1000, Cell Signaling), rabbit anti-E-cadherin (1:1000, Cell Signaling), mouse anti-PPP1R16A (MYPT-3) (1:500, abcam), mouse anti-Protein Phosphatase 1 beta (PP1β) (1:1000, abcam), rabbit anti-Wnt3A (1:1000, Cell Signaling), mouse anti-β-tubulin (1:1000, ABM), rabbit anti-GAPDH (1:3000, Cell Signaling), mouse anti-Na+/K+ ATPase (1:50 DSHB), rabbit anti-Histone H3 (1:1000 Cell Signaling).

Techniques: Luciferase, TOPFlash assay, Activity Assay, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control