Journal:

Article Title: Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction

doi:

Figure Lengend Snippet: Characterization of end-joining activity and immunoreactive polypeptides in heparin–agarose, Q-Sepharose and Superdex 200 fractions. (A) Upper panel: end-joining assays using heparin–agarose column fractions. Assays were performed as described in Materials and Methods, and contained 2 µl of flow-through (FT), 2 µl of fractions eluted with buffer containing KOAc (molar concentration indicated) or 2 µl of nuclear extract (NE). Fractions containing >0.1 M KOAc were subjected to dialysis against DB buffer containing 0.1 M KOAc prior to assay. Reactions were performed in the presence or absence of 100 ng of purified, exogenous DNL IV/XRCC4 complex as indicated (+ or –). Products were resolved by SDS–agarose gel electrophoresis and visualized by PhosphorImager analysis. Lower panels: immunoblotting using heparin–agarose column fractions. Fractions are the same as in the upper panel, except that 10 µl was used. Proteins were resolved by 7.5% SDS–PAGE and transferred to nitrocellulose. Blots were probed with a mixture of anti-DNA-PKcs (mAb 18-2), anti-Ku80 (mAb 111) and anti-Ku70 (mAb N3H10), or with anti-NBS1 (rabbit polyclonal serum, Novus Biologicals, Littleton, CO) as indicated. (B) Upper panel: end-joining assays using Q-Sepharose column fractions. Assays were performed as in (A), and contained 2 µl of fractions eluted with buffer containing KOAc (molar concentrations indicated). Some reactions contained Ku heterodimer (25 ng) as indicated. Lower panel: immunoblotting using Q-Sepharose fractions. Fractions are the same as in the upper panel, except that 15 µl was used. Blots were probed with the same antibodies as in (A). (C) Upper panel: end-joining assays using Superdex 200 fractions. Reactions contained 2 µl of pooled Q-Sepharose 0.85 M fraction or 3 µl of individual Superdex 200 fractions as indicated. Middle panels: immunoblotting using 10 µl of 0.4 M heparin–agarose fraction (HA), 15 µl of 0.85 M Q-Sepharose fraction (QS) or 20 µl of Superdex 200 fractions as indicated. Blots were probed with a mixture of antibodies as in (A). Lower panel: Superdex 200 elution profile showing absorbance trace at 280 nm. Dashed lines indicate region of profile analyzed in activity and immunoblotting assays. Column void volume is indicated (V0).

Article Snippet: Blots were probed with a mixture of anti-DNA-PKcs (mAb 18-2), anti-Ku80 (mAb 111) and anti-Ku70 (mAb N3H10), or with anti-NBS1 (rabbit polyclonal serum, Novus Biologicals, Littleton, CO) as indicated. ( B ) Upper panel: end-joining assays using Q-Sepharose column fractions.

Techniques: Activity Assay, Concentration Assay, Purification, Agarose Gel Electrophoresis, Western Blot, SDS Page

Journal: Experimental and Therapeutic Medicine

Article Title: Negative pressure wound therapy enhances bone regeneration compared with conventional therapy in a rabbit radius gap-healing model

doi: 10.3892/etm.2021.9905

Figure Lengend Snippet: VEGF, BMP-2 and OPN protein expression. (A) Representative western blot image and quantification of (B) VEGF, (C) BMP-2 and (D) OPN protein expression at each time point examined in the NPWT and control groups. * P<0.05 and ** P<0.01. VEGF, vascular endothelial growth factor; BMP-2, bone morphogenetic protein 2; OPN, osteopontin; NPWT, negative pressure wound therapy; 2W, week 2; 4W, week 4.

Article Snippet: The membranes were then incubated overnight at 4˚C with TBS-Tween-20 supplemented with one of the following primary antibodies: Mouse anti-β-actin (1:1,000; Boster Biological Technology; cat. no. P60709), mouse anti-VEGF (1:200; Santa Cruz Biotechnology, Inc. cat. no. sc-7269), mouse anti-BMP-2 (1:400; Santa Cruz Biotechnology, Inc. cat. no. sc-137087) or mouse anti-OPN (1:400; Abcam; cat. no. ab228748).

Techniques: Expressing, Western Blot