atf2 pt71 5112s rabbit monoclonal  (Santa Cruz Biotechnology)

Journal: Biology Open

Article Title: Tpl2 is required for VEGF-A-stimulated signal transduction and endothelial cell function

doi: 10.1242/bio.034215

Figure Lengend Snippet: Tpl2 is required for VEGF-A-stimulated ATF-2-dependent VCAM-1 expression. (A) Control, scrambled, Tpl2 or ATF-2-specific siRNA-treated endothelial cells were stimulated (25 ng/ml) with either VEGF-A 165 or VEGF-A 121 (negative control) for 4 or 8 h, prior to immunoblot analysis. (B–E) Quantification of protein levels by immunoblot analyses (see the Materials and Methods) of VEGF-A (25 ng/ml) stimulated control, scrambled, Tpl2-depleted or ATF-2 depleted endothelial cells for protein levels of (B) VCAM-1, (C) Tpl2, (D) ATF-2-pT71 and (E) ATF-2. Error bars indicate ±s.e.m. ( n =3). In panels B–E, statistical tests=two-way ANOVA; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Antibodies: goat-anti-VEGFR2 cat. #AF357, mouse-anti-VCAM-1 cat. #BBA5 (R&D Systems, Minneapolis, MN, USA), goat-anti-nectin 2 cat. #AF2229 (R&D Systems), rabbit-anti-ATF-2 cat. #35031, rabbit-anti-phospho-ATF-2 cat. #9221 (pT71), rabbit-anti-phospho-VEGFR2-Y1175 (pY1175) cat. #2478 (Cell Signaling Technology, Danvers, MA, USA), goat anti-VE-cadherin, cat. #sc-6458, mouse-anti-PECAM-1 (CD31) cat. #sc-65260, mouse-anti-Tpl2 cat. #37677 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse-anti-PVR (SKII.4) cat. #132202 (BioLegend, San Diego, CA, USA), mouse-anti-α-tubulin, (Sigma-Aldrich, Poole, UK).

Techniques: Expressing, Control, Negative Control, Western Blot

Journal: Biology Open

Article Title: Tpl2 is required for VEGF-A-stimulated signal transduction and endothelial cell function

doi: 10.1242/bio.034215

Figure Lengend Snippet: Tpl2-selective kinase inhibitor treatment attenuates ATF-2 phosphorylation and expression levels. (A–F) Analysis of basal endothelial signaling in non-stimulated cells. (A) Immunoblot analysis of endothelial cells treated with Tpl2-selective kinase inhibitor (2 µM) for 0, 1, 2, 4 or 8 h to assess effects on VEGFR2, ATF-2 and Tpl2 status. Quantification of immunoblot data for Tpl2-selective inhibitor time course for (B) ATF-2-pT71, and (C) Tpl2 levels. Error bars indicate ±s.e.m. ( n =4). In panels B and C, statistical tests=one-way ANOVA; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. (D) Immunoblot analysis of endothelial cell response to Tpl2-selective kinase inhibitor titration (2–50 µM, 4 h). (E,F) Quantification of immunoblot data from Tpl2-selective kinase inhibitor titration on (E) ATF-2-pT71, and (F) ATF-2 levels. Error bars indicate ±s.e.m. ( n =3). In panels E and F, statistical tests=one-way ANOVA; * P <0.05, ** P <0.01. (G) Endothelial cells were pre-treated with a Tpl2-selective kinase inhibitor (2 µM; 30 min) prior to stimulation with either (25 ng/ml) VEGF-A 165 (165) or VEGF-A 121 (121, negative control) for 0 or 8 h before lysis and immunoblot analysis. (H) Quantification of immunoblot data showing changes in relative VCAM-1 levels upon VEGF-A (25 ng/ml) treatment in the absence (−) or presence (+) of Tpl2 kinase inhibitor. Error bars indicate ±s.e.m. ( n =6). Statistical test=two-way ANOVA; * P <0.05, ** P <0.01, **** P <0.0001.

Article Snippet: Antibodies: goat-anti-VEGFR2 cat. #AF357, mouse-anti-VCAM-1 cat. #BBA5 (R&D Systems, Minneapolis, MN, USA), goat-anti-nectin 2 cat. #AF2229 (R&D Systems), rabbit-anti-ATF-2 cat. #35031, rabbit-anti-phospho-ATF-2 cat. #9221 (pT71), rabbit-anti-phospho-VEGFR2-Y1175 (pY1175) cat. #2478 (Cell Signaling Technology, Danvers, MA, USA), goat anti-VE-cadherin, cat. #sc-6458, mouse-anti-PECAM-1 (CD31) cat. #sc-65260, mouse-anti-Tpl2 cat. #37677 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse-anti-PVR (SKII.4) cat. #132202 (BioLegend, San Diego, CA, USA), mouse-anti-α-tubulin, (Sigma-Aldrich, Poole, UK).

Techniques: Phospho-proteomics, Expressing, Western Blot, Titration, Negative Control, Lysis

Journal: Biology Open

Article Title: Tpl2 is required for VEGF-A-stimulated signal transduction and endothelial cell function

doi: 10.1242/bio.034215

Figure Lengend Snippet: Probing the link between Tpl2, Akt activation and ATF-2 status. (A) Immunoblot analysis of endothelial cells titrated with Tpl2-selective kinase inhibitor (2–50 µM, 4 h). Quantification of immunoblot data upon inhibitor titration on activated kinases such as (B) Akt-pS473, (C) ERK1/2-pT202/pY204, (D) p38 MAPK-pT180/pY182 and (E) JNK-pT183/pY185. Error bars indicate ±s.e.m. ( n =3). In panels B–E, statistical tests=one-way ANOVA; * P <0.05. (F) Immunoblot analysis of endothelial cells treated with small molecule kinase inhibitors (2 µM, 30 min) specific for ERK1/2 (PD98059), p38 MAPK (SB203580), JNK (SP600125) and Akt (MK2206). (G) Quantification of immunoblot data for relative ATF-2-pT71 levels upon inhibition of different protein kinases. Error bars indicate ±s.e.m. ( n =3).

Article Snippet: Antibodies: goat-anti-VEGFR2 cat. #AF357, mouse-anti-VCAM-1 cat. #BBA5 (R&D Systems, Minneapolis, MN, USA), goat-anti-nectin 2 cat. #AF2229 (R&D Systems), rabbit-anti-ATF-2 cat. #35031, rabbit-anti-phospho-ATF-2 cat. #9221 (pT71), rabbit-anti-phospho-VEGFR2-Y1175 (pY1175) cat. #2478 (Cell Signaling Technology, Danvers, MA, USA), goat anti-VE-cadherin, cat. #sc-6458, mouse-anti-PECAM-1 (CD31) cat. #sc-65260, mouse-anti-Tpl2 cat. #37677 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse-anti-PVR (SKII.4) cat. #132202 (BioLegend, San Diego, CA, USA), mouse-anti-α-tubulin, (Sigma-Aldrich, Poole, UK).

Techniques: Activation Assay, Western Blot, Titration, Inhibition