Figure 1

ASPL interacts with p97 via the UBX domain. (a) Yeast two-hybrid analyses of p97 using the HIS3 reporter gene. Co-transformation of p97 bait with the indicated p97 binding partner preys supported cell growth under conditions selecting for interaction (in the absence of histidine and the presence of 25 mM 3-aminotriazol (3AT)) (right panel). An empty prey vector served as a negative control. (b) Yeast two-hybrid analyses of ASPL using the HIS3 reporter gene. Co-transformation of ASPL bait with p97 or NSF preys supported cell growth under conditions selecting for interaction (in the absence of histidine and the presence of 25 mM 3-aminotriazol (3AT)) (right panel). An empty prey vector served as a negative control. (c) Purified 6His-tagged p97 was incubated with GST or GST-tagged ASPL and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to p97 (upper panel) Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (d) Purified 6His-tagged NSF was incubated with GST or GST-tagged ASPL and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to the 6His-tag on NSF (upper panels). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). Interaction to NSF was only evident when no detergents were included in the buffer system. In the presence of 0.5% Triton X-100 no interaction between ASPL and NSF was observed. (e) MelJuSo cell lysates were used in immunoprecipitation (IP) experiments with antibodies to ASPL and Protein A Sepharose or as a control Protein A Sepharose beads only. SDS-PAGE and blotting revealed that ASPL co-precipitated p97, but not NSF or the Rpn1 or α subunits of the 26S proteasome.