Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: β-Catenin Preserves the Stem State of Murine Bone Marrow Stromal Cells Through Activation of EZH2

doi: 10.1002/jbmr.3975

Figure Lengend Snippet: β-catenin effects on BMSC differentiation involve EZH2. (A)ChIP-seq for association of β-catenin or TCF4 to promoters of Ezh2 or Ezh1. Overlaid ChIP-seq tracks are displayed as either vehicle (yellow) or LiCl-treated (blue). Overlapping track data appear as green. Genomic location and scale are indicated (top) and maximum height of tag sequence density for each data track is indicated on the y-axis (top left each track, normalized to input and 107 tags). Gene transcriptional direction is indicated by an arrow and exons by boxes. (B) BMSCs cultured in MEM ± LiCl for 48 hours; WB shows EZH2, methylated H3K27, and H3. BMSCs were cultured in A medium for 3 days and were probed for adipocyte markers after overexpression (C) or knockdown of β-catenin (D). (E) BMSCs were cultured in A medium and treated with GSK126 for 3 days (Ezh2i); WB as shown in cells treated with EV or S33Yβcat and for (F) with β-catenin knockdown; see also Fig. S3. (G) BMSCs were cultured in O medium and then treated with GSK126 for 4 days; RT-PCR for osteogenic proteins are shown in response to S33Yβcat overexpression, or in (H) β-catenin knockdown. Graph bars show mean ± SE, *p < .05, **p < .01. EV = empty vector; WB = western blot.

Article Snippet: After blocking, primary antibody was applied overnight at 4°C including antibodies against Pparg, Flag, and EZH2 from Cell Signaling Technology (Beverly, MA, USA; cat #2443, 8146, and 5246, respectively); Histone H3 (Sigma-Aldrich; cat# 05–928), β-catenin (Fisher Scientific; cat# PIPA516762), methylated histone (H3K27me) (Fisher Scientific; 17-622-MI), Adipoq (Fisher Scientific; cat# PA1054), Fabp4 (ProSci, Poway, CA, USA; cat# XG-6174), beta-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat# sc-23949).

Techniques: ChIP-sequencing, Sequencing, Cell Culture, Methylation, Over Expression, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Western Blot

Journal:

Article Title: Recombinant Rinderpest Vaccines Expressing Membrane-Anchored Proteins as Genetic Markers: Evidence of Exclusion of Marker Protein from the Virus Envelope

doi:

Figure Lengend Snippet: Analysis of HA and ANC-GFP expression and secretion by RPVINS-HA- and RPVANC-GFP-infected B95a cells. Cells were labeled for 2 h and then the medium was replaced with normal medium containing unlabeled methionine and cysteine. Medium was collected after 0, 1, 2, 4, 6, and 8 h of chase and analyzed for the presence of labeled secreted proteins, whereas the corresponding cell sheet was lyzed and analyzed for the presence of labeled intracellular proteins. Proteins were immunoprecipitated with either anti-HA polyclonal antibody (αHA), anti-GFP polyclonal antibody (αGFP), or anti-RPV P monoclonal antibody (αRPV-P) as described in Materials and Methods. (A to D) RPVINS-HA-infected cells; (E to H) RPVANC-GFP-infected cells. Numbers above the gels represent the time in hours after protein labeling at which the samples were collected. Positions of the molecular mass markers in kilodaltons are indicated on the left. Positions of the RPV N and P proteins are indicated on the right.

Article Snippet: Antibodies used were 0.5 μl of rabbit anti-HA polyclonal antibody (provided by D. A. Steinhauer), 0.5 μl of polyclonal rabbit anti-GFP antibody (Clontech), and 0.2 μl of mouse anti-RPV P (phosphoprotein) monoclonal antibody 2-1 ( 5 ) together with 0.5 μl of rabbit anti-mouse antibody (Dakopatts).

Techniques: Expressing, Infection, Labeling, Immunoprecipitation