Necdin reduces nuclear PIAS1 levels in transfected cells.

<p>(<b>A</b>, <b>B</b>) Localization of PIAS1 and necdin in transfected cells. HEK293A cells were transfected with expression vectors for Myc-PIAS1 (Myc-PIAS1, 3 µg), necdin (Necdin, 2 µg) or both (Myc-PIAS1+Necdin) and fixed 24 hrs later (<b>A</b>). Transfected cells were treated with 30 µM MG132 for 4 hrs prior to harvest (<b>B</b>). Cells were immunostained with antibodies to Myc (top panels) and necdin (middle panels). Chromosomal DNA (DNA) was stained with Hoechst 33342 (bottom panels). (<b>C, D</b>) Quantification of PIAS1-expressing cells. Fluorescence intensities of PIAS1-expressing cells cotransfected with necdin were classified into three groups; undetectable or barely detectable (±), low (+), and high (++). Arrowheads point to representative cells (<b>C</b>). Classified PIAS1-expressing cells were quantified as % of necdin-expressing cells (<b>D</b>). Transfection efficiency (mean ± SD, <i>n</i> = 3): (MG132-) PIAS1, 24±4%; Necdin, 32±5%; PIAS1+Necdin, 32±5%; (MG132+) PIAS1, 27±1%; Necdin, 33±3%; PIAS1+Necdin, 39±4%. Values (<b>D</b>) represent the mean ± SD (<i>n</i> = 3). *<i>p</i><0.05. Scale bars (<b>A</b>–<b>C</b>), 20 µm.