Fig 1.

SPF45 is a novel ERK2 coimmunoprecipitating substrate. (A) Cell lysates from SKOV-3 clones expressing FLAG-ERK2-Q103G were immunoblotted for ERK2. (B) Cells from clone T8 (designated SKOV-3-QG) from panel A were immunostained with antibodies to FLAG. (C) Anti-FLAG immunoprecipitates from adherent or suspended SKOV-3-QG cells were incubated in an in vitro kinase assay with [γ-³²P]N⁶-cyclopentyl-ATP. The reaction mixtures were run on a gel, silver stained, and processed for autoradiography (right panel). A parallel reaction from six 15-cm dishes of suspended cells was run on the same gel and silver stained (left panel). The arrow indicates the band excised from the gel and processed for mass spectrometry. (D) Myc-SPF45 coimmunoprecipitates with Flag-ERK2. COS-1 cells were transfected with Myc-SPF45 and Flag-ERK2 and Flag immunoprecipitates, and cell lysates were immunoblotted as indicated. (E) Similar to panel D, but Myc immunoprecipitates were immunoblotted. (F) Validation of a novel SPF45 antibody. Either buffer or His-SPF45 was run on a gel and immunoblotted with anti-SPF45 antibody. (G) COS-1 cells were transfected with either empty vector or untagged SPF45 and Myc-SPF45. Cellular lysates were immunoblotted with anti-SPF45 antibody with a short (left panel) or a long (right panel) exposure, the latter of which shows recognition of endogenous SPF45 (lane 3, band next to *). NS, nonspecific band. (H) Partially activated His-ERK2 or buffer was incubated overnight with a nuclear extract from HeLa cells. His-ERK2 was reisolated, run on a gel, and immunoblotted with anti-SPF45 and anti-ERK2 antibody. (I) Same as in panel H, but increasing amounts of His-ERK2 were incubated with the same amount of HeLa nuclear extract per sample.