Figure 2. During cell division, WSTF phosphorylation events govern the assembly and disassembly of B-WICH.

(A) NM1, SNF2h and WSTF are co-precipitated from protein extracts prepared from growing HeLa cells. Bound proteins were detected on immunoblots. 10% of the input is shown in Lane 1. IP, immunoprecipitation. (B) Co-precipitations of NM1, SNF2h and WSTF are impaired from protein extracts prepared from mitotic HeLa cells at t = 0 min from the nocodazole block. Bound proteins were detected on immunoblots. 10% of the input is loaded in Lane 1. IP, immunoprecipitation. (C) WSTF becomes phosphorylated at the onset of mitosis. Lysates were prepared from growing HeLa cells (Lane 1), mitotic HeLa cells at t = 0 min from the nocodazole block (Lanes 2 and 3) or HeLa cells after t = 120 min release from the block (Lanes 4 and 5). Where indicated extracts were subjected to phosphatase treatment (Lanes 3 and 5). In all cases lysates were separated by 7% phospho-affinity SDS-PAGE and analyzed on immunoblots for WSTF and actin. (D) Co-precipitations of WSTF, SNF2h and NM1 are dependent on WSTF phosphorylation. Mitotic extracts from HeLa cells at t = 0 min from the nocodazole block, untreated (Lanes 1–3) or treated with phosphatase (Lanes 4–6), were subjected to immunoprecipitations with anti-WSTF antibodies or non-specific IgGs. Bound proteins were separated by SDS PAGE and analyzed on immunoblots for WSTF, SNF2h and NM1. 5% of the input is shown in Lane 1. IP, immunoprecipitation. (E–F) WSTF steady state expression levels on immunoblots of lysates prepared from control (scrRNAi) and WSTF-silenced HeLa cells. Right panel, densitometric quantification of WSTF steady state protein expression relative to actin. (G) In growing HeLa cells, co-precipitations of NM1 and SNF2h are impaired as a consequence of WSTF gene knockdown by siRNA (WSTF RNAi) but not when cells are subjected to control experiments with scrambled RNAi oligonucleotides (scrRNAi). In Lanes 1 and 4, 20% of the input material was loaded.