Fig. 3. Effects of SKD1(E235Q) on the subcellular localization and oligomer formation of SBP1. (A) Post nuclear supernatant prepared from control and GFPSKD1(E235Q) adenovirus-infected rat 3Y1 fibroblast cells were fractionated into cytosol (sup.) and membrane (ppt.) by centrifugation at 105,000 g for 60 minutes. Comparable amounts of each fraction were processed for immunoblotting with anti-SKD1 and anti-SBP1 antibodies. (B) A cell lysate prepared from HeLa cells with binding buffer was fractionated by size exclusion chromatography on a Superdex 200 column. The collected fractions were processed for immunoblotting with anti-SKD1 and anti-SBP1 antibodies. Positions of molecular mass standards are indicated by arrows. (C) A cell lysate prepared from GFP-SKD1(E235Q) adenovirus-infected HeLa cells was fractionated and analyzed as described in B. (D) Chemical cross-linking of SBP1. NIH3T3 cell lysates were incubated with non-cleavable cross-linker DSS (0, 10, 100 and 1000 µM) for 60 minutes and then processed for immunoblotting with anti-SBP1 antibody.