Journal: Journal of Molecular Signaling
Article Title: Abundance, complexation, and trafficking of Wnt/?-catenin signaling elements in response to Wnt3a
Figure Lengend Snippet: Differential effects of Wnt3a stimulation on Dvl1, Dvl2, and Dvl3 . Panel A , the comparison of the cellular abundance of mammalian Dvl isoforms (Dvl1, Dvl2, and Dvl3) in subcellular fractions. F9 cells expressing Rfz1 were used to prepare subcellular fractions. Plasma membrane-enriched (PM), cytosol-enriched (CY), and nuclear-enriched (NU) fractions were prepared as described in detail in Methods and are displayed. The results of multiple densitometric scans of immunoblots were quantified. The results are displayed based upon the distribution (%), the total protein content of the whole-cell homogenate and fractions, and quantified analysis of blots from SDS-PAGE for each molecule and various controls for sample loading and blotting. The results are shown as mean values ± S.E. from 6–8 independent experiments. Panel B , the comparison of the cellular abundance of Dvls in response to Wnt3a. F9 cells expressing Rfz1 were stimulated with purified Wnt3a for indicated time and then disrupted in a lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na 3 VO 4 , 50 mM NaF, 1 % Triton X-100, 1 mM phenymethysulfonyfluoride (PMSF), 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. Abundance of each Dvl isoform was determined by staining of blots with specific antibodies against each Dvl. Fractions were also stained with anti-GAPDH antibody to establish loading equivalence. Values are displayed as ''fold'' of zero-time point (set to 1). A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 6 independent experiments. Panel C , F9 cells expressing Rfz1 receptor were stimulated without or with Wnt3a. Cells were harvested at each time point indicated, cell lysates were fractionated into plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions. Quantified immunoblotting was performed as described in Methods . Each fraction (100 μg) was subjected to SDS-PAGE and the resolved proteins analyzed by immunoblotting with anti-Dvl1-, anti-Dvl2- and anti-Dvl3-specific antibodies. The three panels displayed at the bottom of the immunoblot set show blots stained with antibodies to well known subcellular marker proteins: Na + -K + -ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. Representative blots are shown ( top panel ). Summary of quantified analysis of blots from SDS-PAGE are shown ( bottom panel ). The results are shown as mean values ± S.E. from 4–6 independent experiments. Dvl1 (blue line), Dvl2 (pink line) and Dvl3 (green line) are displayed.
Article Snippet: Materials The following reagents were purchased from the indicated commercial supplier(s): anti-Dvl1, Dvl2 and Dvl3 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GSK3β antibody from Cell Signaling (Danvers, MA); anti-Axin, anti-protein phosphatase 2A (PP2A) C subunit, and anti-phosphoserine-9-GSK3β antibodies from Upstate Biotechnology (Lake Placid, NY); anti-β-catenin antibody from Sigma (St. Louis, MO); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-Na+ -K+ -ATPase, and anti-fibrillarin antibodies from Abcam (Cambridge, MA); anti-cytokeratin endoA antibody TROMA-1 from the University of Iowa Developmental Studies Hybridoma Bank (Iowa City, IA), Immobilon membrane from Millipore (Bedford, MA) ;and, purified, biologically active Wnt3a and anti-Axin antibody from R & D Systems (Minneapolis, MN).
Techniques: Expressing, Western Blot, SDS Page, Purification, Lysis, Staining, Marker