Figure 3. The TSG101 docking site in the PRD interacts with Patch 2 of the Bro1 domain.
(A) FLAG-TSG101 was ectopically expressed in HEK293 cells. Following cell lysis, cytosolic proteins from cell lysates were immunoprecipitated with polyclonal anti-FLAG antibodies in TBS or TBS supplemented with 1% NP-40. Immunocomplexes were immunoblotted with an anti-ALIX antibody mix (upper) or anti-FLAG monoclonal antibody (lower panel).
(B) FLAG-TSG101 was ectopically expressed in HEK293 cells. Following cell lysis, cytosolic proteins from cell lysates were mixed with GST-ALIX1-746 or ΔBro1 GST-ALIX1-746. FLAG-TSG101 was then immunoprecipitated with polyclonal anti-FLAG antibodies, and input recombinant proteins and immunocomplexes were immunoblotted with anti-GST (upper panel) or anti-FLAG (lower panel) monoclonal antibody.
(C) FLAG-TSG101 was co-expressed with either WT or ΔP2 GFP-ALIX in HEK293 cells. Following cell lysis, cytosolic proteins from cell lysates were immunoprecipitated with anti-FLAG monoclonal antibody, and input proteins and immunocomplexes were immunoblotted with anti-GFP (upper panel) or anti-FLAG polyclonal antibodies (lower panel).
(D) HEK293 cells were transfected with an expression vector for WT or the M95A mutant form (mut) of FLAG-TSG101 or an empty vector as a control. Following cell lysis, cytosolic proteins from cell lysates were immunoprecipitated with each of the indicated anti-ALIX antibodies, and input cytosolic proteins and immunocomplexes were immunoblotted with an anti-ALIX antibody mix (upper panel) or anti-FLAG polyclonal antibodies (lower panel).
(E) The wild type (WT) or TDS-deleted (ΔTDS) FLAG-ALIXPRD was ectopically expressed in HEK293 cells. Following cell lysis, cytosolic proteins from cell lysates were incubated with immobilized GST-ALIXBro1, and input and bound proteins were immunoblotted with anti-FLAG (upper panel) or anti-GST monoclonal antibody (lower panel).
(F) WT or ΔTDS GFP-ALIX was ectopically expressed in HEK293 cells. Following cell lysis, cytosolic proteins from each transfectant were split equally into four samples, which were immunoprecipitated with each of the indicated anti-ALIX antibodies. The immunocomplexes were immunoblotted with anti-GFP monoclonal antibody.
(G) WT or ΔTDS GFP-ALIX was ectopically expressed in HEK293 cells. Following cell lysis, cytosolic proteins were incubated with immobilized GST-p9 or GST-p6, and input and bound proteins were immunoblotted with anti-GFP monoclonal antibody.
(H) FLAG-CHMP4b was ectopically expressed in HEK293 cells. Following cell lysis, cytosolic proteins from cell lysates were immunoprecipitated with anti-FLAG polyclonal antibodies. Then, the FLAG-CHMP4b immunocomplex was incubated with WT or ΔTDS GST-ALIX1–746, and input recombinant proteins and bound proteins were immunoblotted with anti-GST (upper panel) or anti-FLAG monoclonal antibody (lower panel).