Journal: PLoS ONE
Article Title: Mapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3
doi: 10.1371/journal.pone.0071518
Figure Lengend Snippet: A. EGFR intracellular domain (ICD) map and plasmid validation. The intracellular domain (ICD) and C-terminal domain (CTD) of EGFR were fused to the Gal4 DNA binding domain (Gal4DBD). CHOK1 cells were transfected with each construct for 48 hr prior to harvesting WC lysate and fractionation on SDS-PAGE followed by immunoblotting for EGFR and Gal4DBD. EGFRWT vector was transfected into CHOK1 cells as a positive control. α-tubulin was used as a loading control . B. EGFR-CTD contains strong transactivation potential. CHOK1 cells were transfected with EGFR-ICD or EGFR-CTD constructs, UAS-luciferase and Tk- Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 3). The graphs are representative of four independent experiments. C. HER3-ICD map and plasmid validation. The HER3- ICD, juxtamembrane and tyrosine kinase domain (JKD), and CTD were fused to the Gal4DBD. Transfection was performed the same as in 1A and immunoblot analysis was performed for HER3 and Gal4DBD. HER3WT was transfected into CHOK1 cells as a positive control. α-tubulin was used as a loading control. D. HER3-CTD contains strong transactivation potential. CHOK1, H226 R , and SKBr3 cells were transfected with HER3-ICD, HER3-JKD, and HER3-CTD constructs, UAS-luciferase and Tk- Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 3). The graphs are representative of four independent experiments. Luciferase activity was normalized to DNA content, protein content, and the expression of Renilla luciferase in all assays. Luciferase activity detected for each construct was normalized to the Gal4DBD vector control. Data points are represented as mean+/−s.e.m. p<0.05. TM (transmembrane); JM (juxtamembrane); KD (kinase domain).
Article Snippet: For the Gal4 UAS-luciferase assay, cells were transiently transfected with 100 ng plasmid DNA, 1 ug of pGL4.35 Gal4 UAS-luciferase vector (Promega), and 100 ng of pRL-Tk- Renilla vector (Promega).
Techniques: Plasmid Preparation, Binding Assay, Transfection, Construct, Fractionation, SDS Page, Western Blot, Positive Control, Luciferase, Activity Assay, Expressing
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