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. 2006 Apr;26(8):3039–3047. doi: 10.1128/MCB.26.8.3039-3047.2006

FIG. 6.

FIG. 6.

PKA phosphorylates MEKK2 in vitro. (A) HEK293 cells were grown to confluence and cultured in serum-free DMEM for 24 h prior to harvest. Endogenous MEKK2 was immunoprecipitated from cell lysates and used as the substrate in in vitro kinase assays performed with diluent or increasing concentrations of PKA. The autoradiogram (top) and immunoblot of MEKK2 are shown. (B) HEK293 cells were transfected with the following cDNAs: two left lanes, 5 μg of empty vector; two middle lanes, 0.5 μg 3X-FLAG-MEKK2KM; two right lanes, 5 μg 3X-FLAG-MEKK2KM. After 18 h, medium was removed, and cells were cultured for an additional 18 h in serum-free DMEM. 3X-FLAG-MEKK2KM was immunoprecipitated from cell lysates and used as the substrate for in vitro kinase assays performed with PKA. The autoradiogram (top) and immunoblot of 3X-FLAG-MEKK2 are shown. (C) HEK293 cells were transfected with 5 μg cDNA encoding 3X-FLAG-MEKK2KM. After 18 h, cells were transferred to serum-free DMEM for an additional 18 h. 3X-FLAG-MEKK2KM was immunoprecipitated from lysates and used as the substrate for GST-PAK1 (232-454) or PKA. The autoradiogram (top) and immunoblot of 3X-FLAG-MEKK2 (bottom) are shown. Results shown in panels A, B, and C are representative of at least three independent experiments.