FIG. 3.

Lsh is associated with chromatin. (A) Lsh is present in the Triton X-resistant fraction. After Lsh induction in 3T3 cells that were transfected with the Lsh expression vector (Lsh) or transfected with the empty control vector (Ctrl) cells were extracted with Triton X-100, and the resistant and soluble fractions were examined by Western analysis for the presence of Lsh with antibodies against the C-terminal portion of Lsh (Lsh), the Flag epitope (Flag), Brg-1, MeCP2, and PCNA (extracts were prepared from equal number of cells). (B) Kinetics of Lsh expression. At different time points after Lsh induction 3T3 cells were extracted with Triton X, and the different fractions were examined by Western analysis for the presence of Lsh, PCNA, or Vimentin (extracts were prepared from equal number of cells). (C) Association with Triton X-resistant fraction is dependent on cell cycle. 3T3 cells at different stages of confluency were extracted with Triton X, and the different fractions were examined by Western analysis for the presence of Lsh or PCNA and Vimentin serving as controls (extracts were prepared from equal number of cells).(D) The C-terminal and internal domain are required for Triton X resistance. After transfection with the indicated deletion mutants, Triton X-resistant (lanes R) and soluble (Triton flush out [lanes F]) fractions were examined by Western analysis for the presence of GFP, Vimentin, and PCNA (extracts were prepared from equal number of cells).