FIG. 2.

Bag1-L augments c-Jun transcriptional activity. (A) HCT116 cells were transfected with pGL3-TATA-luc, pGL3-uPA-TATA-luc, or pGL3-collagense-TATA-luc, together with c-Jun, Bag1-L, and Ubi-Rluc. Some samples were exposed to UV (100 kJ/m²) for 8 h prior to processing. Luciferase activity was normalized to that for Ubi-Rluc. Data are represented as the means of results for triplicate wells ± standard errors of the means (SEM). Data represent luciferase activity relative to that of pGL3-TATA plus empty vector-transfected cells, which was arbitrarily set to 1. (B) Western blot of cell extracts of HCT116 cells transfected with Bag1-L, which were subjected to 50 μM JNK Inhibitor II (SP600125), 25 ng/ml anisomycin, or UV (100 kJ/m²) and immunoblotted with the indicated antibodies. Note that there is no significant change in mobility of Bag1-L after JNK activation or JNK inhibitor treatment. Overexpression of Bag1-L results in an increase in hyperphosphorylated c-Jun versus that in parental HCT116 cells. Anisomycin and UV robustly increase levels of phospho-c-Jun and JNK inhibitor treatment decreases this substantially versus those in parental HCT116 cells. (C) HEK293T cells were transfected to express Gal4-DBD or Gal4-DBD-c-Jun, Bag1-L or Bag1-S, 5× Gal4UAS-luc, and the Ubi-Renilla-luc transfection control reporter gene (Ubi-Rluc). Luciferase activity was normalized to that with Ubi-Rluc. Data are represented as the means of triplicate wells ± SEM. Activity for Gal4-DBD + empty vector is arbitrarily set to 1. (D) NIH 3T3 cells were transfected to express Gal4-DBD or Gal4-DBD-c-Jun, hBag1-L or NLS-hBag1-S, 5× Gal4UAS-luc, and the Ubi-Renilla-luc transfection control reporter gene (Ubi-Rluc). Luciferase activity was normalized to that for Ubi-Rluc. Data are represented as the means of triplicate wells ± SEM. Activity for Gal4-DBD plus empty vector is arbitrarily set to 1. Targeting Bag1-S to the nucleus does not result in an increase in luciferase reporter activity. (E) NIH 3T3 cells were transfected to express Gal4-DBD-c-Jun or hBag1-L ± NLS-hBag1-S (in either a 1:1 or 2:1 ratio), 5× Gal4UAS-luc, and the Ubi-Renilla-luc transfection control reporter gene (Ubi-Rluc). Luciferase activity was normalized to that for Ubi-Rluc. Data are represented as the means of results for triplicate wells ± SEM. Targeting of Bag1-S to the nucleus and coexpression with Bag1-L result in a significant reduction of luciferase reporter activity.