Figure 5. MEKK2-induces paxillin ubiquitylation.

(A) FLAG-paxillin, HA-MEKK2 and Myc-ubiquitin were co-expressed in 293T cells, and ubiquitylation of immunoprecipitated paxillin is revealed by anti-Myc immunoblot (top panel). Co-immunoprecipitated MEKK2 and total immunoprecipitated paxillin are shown in the second and third panels respectively. HC = Heavy Chain. (B) 293T cells transfected with FLAG-paxillin and HA-MEKK2 were lysed with 2% SDS and boiled to dissociate non-covalent protein interactions. FLAG-paxillin was then immunoprecipitated, and paxillin ubiquitylation was detected by anti-Myc immunoblot (upper panel)whereas the total immunoprecipitated paxillin is shown in the middle panel. MEKK2 expression was confirmed by lysate anti-MEKK2 immunoblot (bottom panel). (C) MDA-MB 231 cells that stably express FLAG-tagged wild type paxillin or LD1-deleted mutant paxillin were lysed and FLAG-tagged wild type or mutant paxillin was immunoprecipitated with anti-FLAG antibodies and separated by SDS-PAGE. Paxillin ubiquitylation was assessed by anti-ubiquitin immunoblot (upper panel), and immunoprecipitation efficiency was confirmed by anti-paxillin immunoblot (second panel). The expression of the tagged paxillin proteins was confirmed by anti-FLAG immunoblot of cell lysates (bottom panel), as was the ability of the anti-ubiquitin antibodies to detect endogenous ubiquitin (third panel). (D) Immunoblots with ubiquitin antibodies that specifically detect anti-K48-linked (upper panel) and anti-K63-linked ubiquitin (second panel) showing linkage of MEKK2-induced ubiquitylation of paxillin immunoprecipitated from transfected cell lysates. The membrane was re-probed with anti-FLAG antibodies (third panel) to demonstrate the effectiveness of the immunoprecipitation, and the bottom panel shows an anti-MEKK2 immunoblot of the cell lysates to confirm MEKK2 expression resulting from the transfected plasmid. The blots are representative of at least three independent experiments.