Figure 1.

Full length coilin cleaved to 28 kDa. fragment in cells. (A) Cleaved coilin fragment levels differ between interphase and mitotic lysates of HeLa and U2OS cells. HeLa and U2OS cells were untreated or treated with nocodazole to achieve mitotic arrest. Interphase and mitotic cell lysates were subjected to SDS-PAGE, Western transfer, and blots probed with anti-coilin and anti-β-tubulin antibodies. (B) Coilin siRNA treatment reduces cleaved coilin fragment levels. HeLa cells were harvested 48 and 72 hr post-transfection with control or coilin-targeted siRNA, lysates were subjected to SDS-PAGE, Western transfer, and blots probed with anti-coilin and anti-β-tubulin antibodies. (C) Cleaved fragment recognized by both poly- and mono-clonal coilin antibodies. U2OS lysates were subjected to SDS-PAGE and Western transfer. The blot was probed with anti-coilin H300 antibodies, followed by stripping and reprobing of the blot with anti-coilin pδ antibodies. (D) Cleaved coilin fragment solubility is different from full length coilin. HeLa cells were untreated or nocodazole treated to achieve interphase (I) and mitotic cells (M). Cells were fractionated as described in methods, and equal volumes of each fraction (DNase I, ammonium sulfate (AS), high salt (HS) and final pellet) were subjected to SDS-PAGE, Western transfer, and blots probed with anti-coilin antibodies (top panel) and anti-acetyalated histone 3 (H3K14) antibodies (bottom panel).