Journal: eLife
Article Title: VGLL2 and TEAD1 fusion proteins identified in human sarcoma drive YAP/TAZ-independent tumorigenesis by engaging EP300
doi: 10.7554/eLife.98386
Figure Lengend Snippet: ( A ) Co-IP assays showing VGLL2-NCOA2 binding to TEAD1 but not YAP 5SA . YAP 5SA -Flag or TEAD1-Flag was co-expressed with VGLL2-NCOA2-HA in HEK293T cells and immunoprecipitated with an anti-HA antibody. ( B ) VGLL2-NCOA2 binds to endogenous TEAD but not YAP/TAZ. Endogenous YAP/TAZ and TEAD proteins in HEK293T cells were detected by anti-YAP/TAZ and panTEAD antibodies, respectively. ( C ) Co-IP assays showing endogenous YAP/TAZ binding to TEAD1 but not TEAD1-NCOA2. TEAD1-Flag or TEAD1-NCOA2-Flag was expressed in HEK293T cells and immunoprecipitated with an anti-Flag antibody. Endogenous YAP/TAZ proteins were detected by anti-YAP/TAZ antibodies. ( D ) The activity of TBS-Luc reporter in HEK293T cells expressing YAP 5SA , VGLL2-NCOA2, or TEAD1-NCOA2, with TEAD inhibitor CP1 (5 μM) treatment or co-expression of TEAD-ENR repressor construct. Data were expressed as mean ± SD. n=3; ****p<0.0001. NS, no significance. ( E ) Schematic representation of TEAD-ENR. TEA DNA-binding domain (TEA) and Engrailed repressor domain (ENR). ( F ) Immunoblot analysis of TEAD-ENR expression in HEK293T cells. ( G ) Co-IP assays showing YAP/TAZ were not essential for VGLL2-NCOA2 binding to endogenous TEADs. VGLL2-NCOA2-HA was expressed in HEK293T cells with or without YAP/TAZ knockdown and immunoprecipitated with an anti-HA antibody. ( H ) Relative mRNA levels of CCN2 , ANKRD1 , and CCN1 in HEK293T cells with YAP/TAZ knockdown and expressing VGLL2-NCOA2 or TEAD1-NCOA2. Data were expressed as mean ± SD. n=3; ****p<0.0001. Figure 2—source data 1. Original western blot membranes corresponding to indicating the relevant bands. The molecular weight markers are indicated. Figure 2—source data 2. Original western blot membranes corresponding to .
Article Snippet: The primary antibodies used in these assays were: GAPDH (cat. 2118, 1:5000, Cell Signaling Technology, Danvers, MA, USA), YAP/TAZ (cat. 8418, 1:1000, Cell Signaling Technology), panTEAD (cat. 13295, 1:1000, Cell Signaling Technology), V5-tag (cat. 13202, 1:1000, Cell Signaling Technology), EP300 (cat. 86377, 1:1000, Cell Signaling Technology), Flag-tag (cat. 2368, 1:1000, Cell Signaling Technology and cat. F9291, 1:1000, Sigma), and HA-tag (cat. 3724 and cat. 2367, 1:1000, Cell Signaling Technology).
Techniques: Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Activity Assay, Expressing, Construct, Western Blot, Knockdown, Molecular Weight
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