![Fig. 1. A-kinase phosphorylates p45 on Ser169 in vitro and in vivo. (A) BHK cells were transfected with increasing amounts of expression vector encoding wild-type p45 [lanes 1 to 3, 0.3 μg, 0.6 μg, and 1.2 μg of pMT2-p45 (wt), respectively] or mutant (Ser169 → Ala)p45 [lanes 4 to 6, 0.3 μg, 0.6 μg, and 1.2 μg of pMT2-p45 (mut), respectively]. Cell extracts were subjected to Western blotting with a p45-specific antibody as described in Materials and Methods. The p45 doublet, which is thought to result from alternative usage of two translational start sites,50is indicated by a double arrow. (B) BHK cells were transfected with expression vectors encoding wild-type p45 [1.2 μg and 0.4 μg of pMT2-p45(wt), lanes 1 and 2, respectively] or mutant p45 [1.2 μg and 0.4 μg of pMT2-p45(mut Ser169 → Ala), lanes 3 and 4, respectively]; lane 5 shows mock-transfected cells. Cell extracts were subjected to immunoprecipitation with a p45-specific antibody; immunoprecipitates were incubated with [γ-32PO4]ATP and purified C-subunit of A-kinase and applied to SDS-PAGE/autoradiography as described in Materials and Methods. (C) BHK cells were transfected with expression vectors encoding wild-type p45 [1.2 μg of pMT2-p45(wt), lanes 1, 2, and 8] or mutant p45 [1.2 μg of pMT2-p45(mut Ser169 → Ala), lanes 4 and 5]; lanes 3 and 6 show cells transfected with 0.2 μg of wild-type or mutant p45 vector, respectively, and lane 7 shows mock-transfected cells. Cells were incubated with 32PO4 and some cultures were treated with 1 mmol/L 8-Br-cAMP (lanes 2, 3, 5, and 6); cell extracts were subjected to immunoprecipitation with p45-specific antibody (lanes 1 to 7) or control rabbit serum (lane 8) and immunoprecipitates were applied to SDS-PAGE/autoradiography as described in Materials and Methods. (D) Wild-type MEL cells (lanes 1 to 3) and A-kinase–deficient MEL cells (lanes 4 to 6) were incubated with32PO4 and some cultures were treated with 1 mmol/L 8-Br-cAMP (lanes 3 and 5); cell extracts were subjected to immunoprecipitation with p45-specific antibody (lanes 2 to 5) or control rabbit serum (lanes 1 and 6) as described in (C).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/91/9/10.1182_blood.v91.9.3193/3/blod40916001w.jpeg?Expires=1722710340&Signature=Fre9hBMZJBcepwClZtlgz77NSwEaUyNhp~~NR6KXWi4zzdt3GecT7gOzaQTepvTEIMcz3DAzI~qqeR~Ihvd4IkQq2zrJagY9WsRlyOw55sdJ~4zxrDXCDytBHV2bg6ArmX2N372I6LThhMXb6gyEwozlwxUR5~DE23MOCQ0h9xHUkk7W12TS3WkGPUHdcYvLrmV0XW8kd1qerVMC6wwmk~XPaUi6A-difvPPrX~a5ioaHrB2hN-yVo2-ciABQBt0ilH3GpaFLJCYSXHN7qA9kKWTtswSAZzISRnl0xDM4rTIUEG~o24d~uqqKGPMDFDCvHOeH-f7JvsUsZz2ejP2BA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
A-kinase phosphorylates p45 on Ser169 in vitro and in vivo. (A) BHK cells were transfected with increasing amounts of expression vector encoding wild-type p45 [lanes 1 to 3, 0.3 μg, 0.6 μg, and 1.2 μg of pMT2-p45 (wt), respectively] or mutant (Ser169 → Ala)p45 [lanes 4 to 6, 0.3 μg, 0.6 μg, and 1.2 μg of pMT2-p45 (mut), respectively]. Cell extracts were subjected to Western blotting with a p45-specific antibody as described in Materials and Methods. The p45 doublet, which is thought to result from alternative usage of two translational start sites,50is indicated by a double arrow. (B) BHK cells were transfected with expression vectors encoding wild-type p45 [1.2 μg and 0.4 μg of pMT2-p45(wt), lanes 1 and 2, respectively] or mutant p45 [1.2 μg and 0.4 μg of pMT2-p45(mut Ser169 → Ala), lanes 3 and 4, respectively]; lane 5 shows mock-transfected cells. Cell extracts were subjected to immunoprecipitation with a p45-specific antibody; immunoprecipitates were incubated with [γ-32PO4]ATP and purified C-subunit of A-kinase and applied to SDS-PAGE/autoradiography as described in Materials and Methods. (C) BHK cells were transfected with expression vectors encoding wild-type p45 [1.2 μg of pMT2-p45(wt), lanes 1, 2, and 8] or mutant p45 [1.2 μg of pMT2-p45(mut Ser169 → Ala), lanes 4 and 5]; lanes 3 and 6 show cells transfected with 0.2 μg of wild-type or mutant p45 vector, respectively, and lane 7 shows mock-transfected cells. Cells were incubated with 32PO4 and some cultures were treated with 1 mmol/L 8-Br-cAMP (lanes 2, 3, 5, and 6); cell extracts were subjected to immunoprecipitation with p45-specific antibody (lanes 1 to 7) or control rabbit serum (lane 8) and immunoprecipitates were applied to SDS-PAGE/autoradiography as described in Materials and Methods. (D) Wild-type MEL cells (lanes 1 to 3) and A-kinase–deficient MEL cells (lanes 4 to 6) were incubated with32PO4 and some cultures were treated with 1 mmol/L 8-Br-cAMP (lanes 3 and 5); cell extracts were subjected to immunoprecipitation with p45-specific antibody (lanes 2 to 5) or control rabbit serum (lanes 1 and 6) as described in (C).
