Bag101 and Bag102 interact with 26S proteasomes and Hsp70.
2014-01-30T02:45:57Z (GMT) by
<p>(A) Domain organization (shown to scale) of human BAG-1S and the <i>S. pombe</i> homologs Bag101 and Bag102. The truncations used for the precipitation experiments are shown. (B) The indicated GST fusion proteins were used in pull down experiments with extract from <i>S. pombe</i> cells expressing ZZ-tagged Rpn11. The precipitated material was analyzed by SDS-PAGE and blotting for Hsp70 (upper panel), the ZZ-tagged 26S proteasome subunit Rpn11, and 20S particle α subunits (middle panels). Equal loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (C) Immunoprecipitates from wild type <i>S. pombe</i> cells expressing Flag-tagged Bag101 and Bag102 were resolved by SDS-PAGE and analyzed by blotting, using antibodies to the proteasome subunit Mts4/Rpn1, and Flag. (D) Differential interference contrast (DIC) and fluorescence micrographs of wild type <i>S. pombe</i> transformed to express GFP-tagged Bag101 and Bag102. DAPI staining was used to mark the nucleus. (E) Lysates from <i>S. pombe</i> cells transformed to express Flag- and GFP-tagged Bag102 were separated into a soluble fraction and pellet. The pellet fraction was then treated with proteinase K and Triton X-100 as indicated, before the samples were analyzed by SDS-PAGE and blotting. Dph1 served as a control for a soluble protein. Bip1 served as a control for an ER luminal protein.</p>