expression construct for human fl vcp/p97 cloned into pet15b  (Hemmo Pharmaceuticals)

Journal: BMC Cell Biology

Article Title: Human ASPL/TUG interacts with p97 and complements the proteasome mislocalization of a yeast ubx4 mutant, but not the ER-associated degradation defect

doi: 10.1186/1471-2121-15-31

Figure Lengend Snippet: 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.

Article Snippet: The expression constructs for human p97 were kindly provided by Prof. Hemmo H. Meyer (Zürich, Switzerland).

Techniques: Transformation Assay, Binding Assay, Plasmid Preparation, Negative Control, Purification, Incubation, SDS Page, Staining, Immunoprecipitation, Control

Journal: BMC Cell Biology

Article Title: Human ASPL/TUG interacts with p97 and complements the proteasome mislocalization of a yeast ubx4 mutant, but not the ER-associated degradation defect

doi: 10.1186/1471-2121-15-31

Figure Lengend Snippet: ASPL interacts with the p97 N-domain. (a) Schematic diagram of the ASPL domain organization and the various truncations used in the precipitation experiments. (b) Purified 6His-tagged p97 was incubated with GST or the indicated GST-tagged ASPL truncations and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to the 6His-tag on p97 (upper panel). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (c) Schematic diagram of the p97 domain organization and the various truncations used in the precipitation experiments. (d) Purified 6His-tagged p97 and p97 truncations were incubated with GST and GST-tagged ASPL before precipitation and analysis by SDS-PAGE and blotting using antibodies specific for the 6His-tagged p97 proteins (upper panel). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel).

Article Snippet: The expression constructs for human p97 were kindly provided by Prof. Hemmo H. Meyer (Zürich, Switzerland).

Techniques: Purification, Incubation, SDS Page, Staining