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Fig. 5. IKAP co-purifies with various cytosolic proteins. (A) Western blot analysis of equal amounts of proteins from cytosolic and nuclear fractions of cIKAP-strep-expressing HEK293 cells. Anti-IKAP antibody was used to detect endogenous IKAP and cIKAP-strep. Western blot analysis of p150 and GRP75 were used to control the purity of the nuclear and cytosolic extracts, respectively. (B,C) One-STrEP-tag purification. HEK293 cells transiently transfected with either empty vector or cIKAP-strep were harvested 2 days after transfection and cytosolic extracts were prepared as described in the Materials and Methods. Purification was performed according to the manufacturer's instructions (IBA). Eluates were concentrated and run on 10% SDS-PAGE. (B) Western blot analysis of cIKAP-strep detected by anti-IKAP antibody in different purification steps. IP, input; FT, flow through. (C) Silver staining of the purified proteins. The indicated proteins were identified by MALDI-TOF-MS. DNPK1, DNA-dependent protein kinase; USP9X, ubiquitin-specific processing protease. (D) Association of IKAP with its C-terminus. HEK293 cells were transiently transfected with either empty vector, or with FD-IKAP-His or cIKAP-His constructs. After 48 hours, cytosolic extracts were prepared and immunoprecipitated with anti-His antibody. Immunoprecipitates were run on SDS-PAGE and analyzed for endogenous IKAP and the His-tagged IKAP fragments.
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