Fig. 4

E2 decreases Sp1 protein stability in lung cancer cells. Samples of A549 and E2-A549 cells with or without cycloheximide treatment were used to study the indicated proteins by immunoblot analysis (A, B). Samples of A549 and A549-T24 cells with or without cycloheximide treatment were used to study the Sp1 level by immunoblot analysis (C). A549 cells were harvested to perform the immunoprecipitation (IP) experiments with anti-Sp1 antibodies; in addition, the ubiquitination of Sp1 was studied by Western blotting with anti-ubiquitin antibodies (D). Samples of A549 and A549-T24 cells were used to study the levels of Sp1 and ubiquitination signals by Western blotting with anti-Sp1 and anti-ubiquitin antibodies (E). Samples of A549 and E2-treated A549 cells were used to study the mRNA and protein levels of SUMO-1, RNF4 and ubiquitination signals by q-PCR and Western blotting with anti-Sp1, anti-SUMO-1, anti-RNF4 and anti-ubiquitin antibody (F, G). The results of three independent experiments were quantitated, and statistical analysis was performed with a t test; *p < 0.05, **p < 0.01, ***p < 0.005