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Fig. 2. Physical interaction of endogenous IIGP and mHk3. Immunoprecipitation of endogenous IIGP or mHk3 from lysates of IFN{gamma}-stimulated or non-stimulated BMM. (A) The lysates were pre-cleared by immunoprecipitation with isotype matched control mAbs. Subsequently, IIGP was immunoprecipitated with the anti-IIGP mAb 5D9 either prior or after incubation for 30 min at 32°C with addition of GTP{gamma}S or GDPßS. Purified proteins were subjected to SDS-PAGE and western blotting. Co-immunoprecipitated mHk3 was revealed with an affinity-purified rabbit anti-hook3 serum. (B) The lysates were pre-cleared by use of a non-immune rabbit serum and mHk3 was subsequently purified from lysates of BMM stimulated with IFN{gamma} or not. IIGP was revealed on western blots using the anti-IIGP mAb 5D9. To determine the total amount of IIGP (B) or mHk3 (A) recovered by specific immunoprecipitation, 1/5 of the samples that were probed for co-purification were analyzed in parallel by western blotting with the respective antibodies.





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