Figure 4

NHR2 and amino terminal domain of ETO are involved in interaction with hSIN3B. Lysates were prepared from COS-7 cells transfected with hSIN3B (S) in combination with ETO (E) and ETO mutants lacking NHR1 (N1), NHR2 (N2), NHR3 (N3), NHR4 (N4) or ETO lacking 30 amino-terminal aminoacids (30) as described in Material and Methods. (A) IP was performed with α-SIN3B followed by Western blotting with α-ETO (α-E). The ETO mutant lacking NHR2 was not pulled down by hSIN3B (lane 3). (B) Both IP and Western blotting with α-SIN3B (α-S) confirmed expression of hSIN3B (arrowhead). (C) Western blotting performed with α-E demonstrated expression of all the mutants (arrowhead). (D) IP was performed with α-SIN3B or α-ETO followed by Western blotting. The presence in the left panel of ETO in lane 1 but not in lane 4 (lacking 30 amino-terminal aminoacids) showed that the ETO amino-terminus is required for interaction with hSIN3B. Reciprocal experiment showed that hSIN3B co-precipitated ETO (right panel, lane 2) but not ETO – 30 aminoacids (right panel, lane 5). Arrowheads show the position of ETO, ETO – 30 aas and hSIN3B. Lower left and right panels show input of ETO and hSIN3B in 2% of IP lysate. The positions of the molecular weight markers are indicated at the left.