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Fig. 4. pSmad2 is expressed in SP and DN thymocytes. Thymocytes, magnetically separated into subpopulations based on CD4CD8 and CD25CD44 expression, were stained with 1 mg of pSmad2 or Smad2 antibodies followed by ¯ow cytometric analysis. (A) Expression of pSmad2 (purple line) compared to isotype control antibody (green line) within each thymic subpopulation. The pSmad2 antibody was competed with 30-fold molar excess of pSmad2 peptide (red line) and analyzed by FACS. The mean ¯uorescent intensity of pSmad2 (B) and Smad2 (C) cells within the SP, DP and DN cells with respect to their CD25CD44 expression is shown. (D) Percentage ligand-induced increase of mean ¯uorescence intensity of pSmad2 after in vitro stimulation with TGF-b1 (black bars) and <t>ActivinA</t> (hollow bars), compared to mean ¯uorescence intensity of pSmad2 in unstimulated cells, within the SP, DP and magnetically sorted DN cells with respect to their CD25CD44 expression is shown. Ligand-induced pSmad2 expression was calculated according to the formula: 100 3 [(pSmad2 MFIafter stimulation ± isotype MFIafter stimulation)/(pSmad2 MFIbefore stimulation ± isotype MFIbefore stimulation)]. Statistical analysis was carried out with Student's t-test: ** indicates 0.01 < P > 0.001. One out of three similar experiments is shown.
Activina, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Fig. 4. pSmad2 is expressed in SP and DN thymocytes. Thymocytes, magnetically separated into subpopulations based on CD4CD8 and CD25CD44 expression, were stained with 1 mg of pSmad2 or Smad2 antibodies followed by ¯ow cytometric analysis. (A) Expression of pSmad2 (purple line) compared to isotype control antibody (green line) within each thymic subpopulation. The pSmad2 antibody was competed with 30-fold molar excess of pSmad2 peptide (red line) and analyzed by FACS. The mean ¯uorescent intensity of pSmad2 (B) and Smad2 (C) cells within the SP, DP and DN cells with respect to their CD25CD44 expression is shown. (D) Percentage ligand-induced increase of mean ¯uorescence intensity of pSmad2 after in vitro stimulation with TGF-b1 (black bars) and ActivinA (hollow bars), compared to mean ¯uorescence intensity of pSmad2 in unstimulated cells, within the SP, DP and magnetically sorted DN cells with respect to their CD25CD44 expression is shown. Ligand-induced pSmad2 expression was calculated according to the formula: 100 3 [(pSmad2 MFIafter stimulation ± isotype MFIafter stimulation)/(pSmad2 MFIbefore stimulation ± isotype MFIbefore stimulation)]. Statistical analysis was carried out with Student's t-test: ** indicates 0.01 < P > 0.001. One out of three similar experiments is shown.

Journal: International immunology

Article Title: Transforming growth factor-beta- and Activin-Smad signaling pathways are activated at distinct maturation stages of the thymopoeisis.

doi: 10.1093/intimm/dxg139

Figure Lengend Snippet: Fig. 4. pSmad2 is expressed in SP and DN thymocytes. Thymocytes, magnetically separated into subpopulations based on CD4CD8 and CD25CD44 expression, were stained with 1 mg of pSmad2 or Smad2 antibodies followed by ¯ow cytometric analysis. (A) Expression of pSmad2 (purple line) compared to isotype control antibody (green line) within each thymic subpopulation. The pSmad2 antibody was competed with 30-fold molar excess of pSmad2 peptide (red line) and analyzed by FACS. The mean ¯uorescent intensity of pSmad2 (B) and Smad2 (C) cells within the SP, DP and DN cells with respect to their CD25CD44 expression is shown. (D) Percentage ligand-induced increase of mean ¯uorescence intensity of pSmad2 after in vitro stimulation with TGF-b1 (black bars) and ActivinA (hollow bars), compared to mean ¯uorescence intensity of pSmad2 in unstimulated cells, within the SP, DP and magnetically sorted DN cells with respect to their CD25CD44 expression is shown. Ligand-induced pSmad2 expression was calculated according to the formula: 100 3 [(pSmad2 MFIafter stimulation ± isotype MFIafter stimulation)/(pSmad2 MFIbefore stimulation ± isotype MFIbefore stimulation)]. Statistical analysis was carried out with Student's t-test: ** indicates 0.01 < P > 0.001. One out of three similar experiments is shown.

Article Snippet: Recombinant TGF-b1 (2 ng/ml) or ActivinA (2 ng/ml) (R & D Systems, Minneapolis, MN) was added to the cells followed by incubation at 37°C water bath for 5, 30 or 60 min.

Techniques: Expressing, Staining, Control, In Vitro