Sp1–SCL complex interaction.
Sp1 physically interacts with multiple elements of the SCL complex. (A) Sp1 interacts in vitro with SCL, GATA-1, LMO2, and Ldb-1. Pull-down assays were performed with the use of immobilized GST and GST-Sp1, and35S-labeled SCL, GATA-1, LMO2, Ldb-1, E47, and luciferase. Inputs (lane 1) represent 10% of the amounts used in lanes 2 and 3. Coomassie blue–stained GST and GST-Sp1 are shown in lanes 4 and 5. (B) Sp1 specifically interacts with the bHLH domain of SCL. SCL mutants were labeled with 35S-methionine and used in pull-down assays as described in panel A. Protein signals were quantified by means of ImageQuant software (Molecular Dynamics). Binding efficiency (percentage of input) was calculated by comparison with input samples (10%) after subtraction of background GST signals. (C) In vivo interaction between Sp1 and SCL. A 2-hybrid assay was performed by transiently transfecting NIH 3T3 cells with the 5XUAS-tk109-Luc reporter (1.5 μg) and Gal4 DBD-Sp1 (10 ng), Gal4 DBD-E2A (10 ng), and Gal4 AD-SCL (1 μg) expression vectors. Results are shown as fold activation over 5XUAS-tk109-Luc transfected alone, are the mean ± SD of replicate determinations, and are representative of 3 experiments.
