Figure 3. Sin3B, Sin3A and c-Myc influence Mad4 stability. (A) Sin3A or Sin3B expression was silenced in four GBM cell lines by siRNA and c-Myc, Mad4 and β-actin expression was determined by immunoblotting. (B) SF767 cells were transfected with siRNA to knock down c-Myc expression. After 3 d, protein expression was assessed by immunoblotting. (C) Cells were transfected with siRNA oligos (30 pmol/well in 24-well plates) to knock down expression of Sin3B. After 2 d, cells were transfected with Flag-Mad4 plasmids at 2 μg per well. After 24 h, cells were assessed by immunoblotting. (D) Upper panel: SF767, U251 or U373 cells were transfected with 4 μg of Flag-Sin3A or c-Myc plasmid and 2 μg of Flag-Mad4 plasmid. After 48 h, the cell lysates were assessed by immunoblotting. Lower panel: Cells from four cell lines were lysed and assessed by immunoblotting as indicated. (E) SF676 cells were transfected with siRNA to knock down Sin3A and Sin3B expression and protein expression was assessed as indicated. (F) Left, expression of Sin3B in Sin3B wild-type or knockout MEFs; right, Sin3B wild-type or knockout MEFs were transfected with siRNA to deplete Sin3A expression and Mad4 expression was measured by immunoblotting. (G) U373 cells were transfected with siRNA to knockdown expression of Sin3A and Sin3B (AB) or Sin3A, Sin3B and c-Myc (ABM) (20 pmol each in 24-well plates) and protein expression level was determined as indicated. (H) TGR1 (c-Myc+/+) or HO15.19 (c-Myc−/−) cells were transfected with siRNA to silence expression of both Sin3A and Sin3B and the expression of Mad4 protein was assessed by immunoblotting.