Figure 3.

Smyd2 associates with the Sin3 repression complex and is involved in transcriptional repression. (A) Expression of GAL4-Smyd2 fusion protein in 293T cells. Exponentially grown 293T cells were transfected with the constructs indicated and, 48 hours post transfection, whole cell lysate was prepared using RIPA buffer and subjected to western analysis using antibodies directed against the GAL4 tag. A reactive band was detected at the appropriate molecular weight (approximately 66 kD). Extracts from cells, transfected with the GAL4-DBD construct [17], served as negative control. (B) Smyd2 represses transcription of a luciferase reporter. Top panel: Schematic illustration of the reporter construct used. Bottom panel: 10T1/2 cells were transiently co-transfected with the 5XGAL4-SV40-luciferase reporter (1 μg) together with GAL4-DBD or GAL4-Smyd2 (2 μg each). Percent activity of the luciferase was determined in relation to GAL4-DBD. Smyd2 significantly represses the transcription of a luciferase reporter in 10T1/2 cells. (C) Smyd2 associates with HDAC1. Exponentially grown 293T cells were transiently co-transfected with GAL4-DBD or GAL4-Smyd2, together with Flag tagged HDAC1 (HDAC1-F). Whole cell RIPA extracts were immunoprecipitated using an anti-GAL4 antibody and immunoblots were probed with an anti-FLAG antibody. As shown here, Smyd2 associates with HDAC1. RIPA whole cell extracts from GAL4-DBD transfected cells [17] served as negative control. Equal protein amounts in the immunopreciptation assays was demonstrated by analysis of 5% input using anti Flag antibodies. (D) Smyd2 interacts with the Sin3A but not the NuRD complex. Exponentially grown 293T cells were transfected with the constructs indicated and, 48 hours post transfection, whole RIPA lysate was prepared. Antibodies directed against GAL4 were used for immunoprecipitation, followed by western analysis using the antibodies indicated. Smyd2 associates with HDAC1 and Sin3A but not with the components of the NuRD complex, MBD3 or MTA2.