number 4060  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: Estrogen receptor β–dependent Notch1 activation protects vascular endothelium against tumor necrosis factor α (TNFα)-induced apoptosis

doi: 10.1074/jbc.M117.790121

Figure Lengend Snippet: Role of E2 and Notch1 on TNFα regulation of Akt phosphorylation in HUVECs. A and B, Western blotting and densitometry of HUVECs treated with TNFα (10 ng/ml) for 1, 2, or 24 h in the presence of E2 (1 nm). Cells treated with DMSO were used as control (CTRL). Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744), pAkt (Ser-473), and total Akt antibodies. β-Actin antibody was used to ensure equal loading. B, graphs show protein levels after the indicated treatments normalized to untreated control levels, after signal comparison to β-actin expression. Phosphorylated Akt was normalized to the total Akt level. Results are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (comparison between TNFα treatment and corresponding control); °, p < 0.05; °°, p < 0.01; °°°, p < 0.001 (pairwise comparison between plus or minus E2). C and D, Western blotting and densitometry of HUVECs treated with TNFα (10 ng/ml) for 1, 2, or 24 h in the presence of DAPT (5 μm). Cells treated with DMSO were used as control (CTRL). Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744), pAkt (Ser-473), and total Akt antibodies. β-Actin antibody was used to ensure equal loading. Graphs show protein levels after the indicated treatments normalized to untreated control levels, after signal comparison to β-actin expression. Phosphorylated Akt was normalized to the total Akt level. Results are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001 (comparison between TNFα treatment and corresponding control); °, p < 0.05; °°°, p < 0.001 (pairwise comparison between plus or minus DAPT). E and F, Western blotting and densitometry of HUVECs treated with TNFα (10 ng/ml) for 1, 2, or 24 h in the presence of E2 (1 nm) or E2 (1 nm)/DAPT (5 μm). Cells treated with DMSO were used as control. Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744), pAkt (Ser-473), and total Akt antibodies. β-Actin was used to ensure equal loading. Graphs show protein levels after the indicated treatments were normalized to untreated control levels, after signal comparison to β-actin expression. Phosphorylated Akt was normalized to the total Akt level. Results are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (comparison between TNFα treatment and corresponding control); °, p < 0.05; °°°, p < 0.001 (pairwise comparison between E2 and E2 plus DAPT treatment).

Article Snippet: Rabbit polyclonal to estrogen receptor β antibodies (catalog number 5513), rabbit monoclonal to cleaved Notch1 valine 1744 (catalog number 4147), rabbit monoclonal to pAkt (Ser-473 D9E, catalog number 4060), and rabbit polyclonal to Akt (catalog number 9272) were from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Estrogen receptor β–dependent Notch1 activation protects vascular endothelium against tumor necrosis factor α (TNFα)-induced apoptosis

doi: 10.1074/jbc.M117.790121

Figure Lengend Snippet: Effect of wortmannin on TNFα-induced apoptosis in HUVECs. A, HUVECs were treated for 24 h with wortmannin (100 nm). Lysates were electrophoresed and immunoblotted with pAkt (Ser-473) and total Akt antibodies. β-Actin antibody was used to ensure equal loading. B, HUVECs treated for 24 h with DMSO (CTRL), 17β-estradiol (E2, 1 nm), and TNFα (10 ng/ml) in the presence or absence of wortmannin (100 nm), were stained with Annexin V and PI and then cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. C, percentage of apoptotic cells (ratio of Annexin V-positive cells/total cells) is shown. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001.

Article Snippet: Rabbit polyclonal to estrogen receptor β antibodies (catalog number 5513), rabbit monoclonal to cleaved Notch1 valine 1744 (catalog number 4147), rabbit monoclonal to pAkt (Ser-473 D9E, catalog number 4060), and rabbit polyclonal to Akt (catalog number 9272) were from Cell Signaling Technology (Beverly, MA).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Estrogen receptor β–dependent Notch1 activation protects vascular endothelium against tumor necrosis factor α (TNFα)-induced apoptosis

doi: 10.1074/jbc.M117.790121

Figure Lengend Snippet: Effect of PHTPP on TNFα-induced apoptosis, Notch1 activation, and Akt phosphorylation in HUVECs. A, HUVECs treated for 24 h with DMSO (CTRL), E2 (1 nm), PHTPP (1 μm), and TNFα (10 ng/ml) alone and in combination, were stained with Annexin V and PI and then cytometric analysis was performed. Representative Annexin V-PI plots are shown for each treatment. B, percentage of apoptotic cells (ratio of Annexin V-positive cells/total cells) following treatment is shown. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001. C, Western blotting analysis of HUVECs treated with DMSO (CTRL), 17β-estradiol (E2,1 nm), PHTPP (1 μm), and TNFα (10 ng/ml). Lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val-1744), pAkt (Ser-473), and total Akt antibodies. β-Actin antibody was used to ensure equal loading. D, densitometric analysis. Graphs show protein levels after the indicated treatments were normalized to untreated control levels, after signal comparison to β-actin expression. Phosphorylated Akt was normalized to the total Akt level. Results are expressed as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01 (comparison between treatments and corresponding control); °°, p < 0.01; °°°, p < 0.001 (pairwise comparison between plus or minus TNFα treatment).

Article Snippet: Rabbit polyclonal to estrogen receptor β antibodies (catalog number 5513), rabbit monoclonal to cleaved Notch1 valine 1744 (catalog number 4147), rabbit monoclonal to pAkt (Ser-473 D9E, catalog number 4060), and rabbit polyclonal to Akt (catalog number 9272) were from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Staining, Western Blot, Expressing