Journal: Development (Cambridge, England)

Article Title: Stat3-Efemp2a modulates the fibrillar matrix for cohesive movement of prechordal plate progenitors.

doi: 10.1242/dev.104885

Figure Lengend Snippet: Fig. 5. The interaction between Efemp2a and soluble FN or LM. (A) The specific bands of the FN and LM on the gel. (B-U) Efemp2a and its mutant C350 could interact with FN and LM in the supernatant (D,E,I,J) but not in the deposition (Q,S). AD293 cells were infected with adenoviruses expressing GFP, Efemp2a- GFP or C350-GFP, respectively, and the media were collected after 48 h and co-incubated with (B-K) or without soluble FN or LM (M-O). Then, the supernatant (B-O) and deposition (P-U) were separated by centrifugation. The supernatant underwent co-immunoprecipitation with anti-GFP (B,D,G,I,M), FN (C,E,N) or LM (H,J,O) antibodies or IgG (F,K). F and K show the negative controls for E and J. (L) The OD ratios of GFP, Efemp2a or C350 bands (E,J) to negative controls (F,K). FN or LM deposition was formed in DMEM without GFP, Efemp2a or C350 in T. T is the negative control for Q and S. (U) The OD ratios of GFP, Efemp2a or C350 bands (Q,S) to negative controls (T). The OD value was defined as 1 when no specific bands were observed in gels. Data were derived from three independent experiments (L,U). #P>0.05, **P<0.01 versus GFP group; Student’s t-test.

Article Snippet: The 10 ml of harvested culture medium was mixed with 20 μl Protein A/G agarose beads and 2 μg anti-GFP antibody (Novus, NBP1-47584; 1:5000).

Techniques: Mutagenesis, Infection, Expressing, Incubation, Centrifugation, Immunoprecipitation, Negative Control, Derivative Assay

Journal: Development (Cambridge, England)

Article Title: Stat3-Efemp2a modulates the fibrillar matrix for cohesive movement of prechordal plate progenitors.

doi: 10.1242/dev.104885

Figure Lengend Snippet: Fig. 6. Efemp2a modulates the self-assembly of FN and LM in vitro. (A) Agarose beads absorb fluorescent proteins via immunoprecipitation. Adenoviruses overexpressing GFP, Efemp2-GFP or C350- GFP were used to infect AD293 cells and the cell lysates were collected. Samples were incubated with Protein A/G beads and anti-GFP antibody. Scale bars: 200 µm. (B) Efemp2a activity modulates the self-assembly of FN and LM into fibers in vitro. Soluble FN (a-c) or LM (e-g) spontaneously assembled into fibers in the presence of GFP (a,e), Efemp2a (b,f) or C350 (c,g) beads. The arrowheads indicate the branches in a-c; the widths of the regions labeled by arrows are measured in e-g. Quantification of the branch numbers of FN fibers (d) or the mean widths of LM fibers (h). Scale bars: 20 µm. (C) Efemp2a activity affects the polymerization and deposition of FN and LM. FN-Rhodamine (a-c) or LM-Rhodamine (d-f) deposition by self-assembly. Quantification of the mean density of FN-Rhodamine or LM-Rhodamine deposition (g,h). *P<0.05, **P<0.01 versus control; Student’s t-test.

Article Snippet: The 10 ml of harvested culture medium was mixed with 20 μl Protein A/G agarose beads and 2 μg anti-GFP antibody (Novus, NBP1-47584; 1:5000).

Techniques: In Vitro, Immunoprecipitation, Incubation, Activity Assay, Labeling, Control