Fig. 8. In vivo and in vitro phosphorylation of ank3 isoforms from MDCK cells. A whole cell lysate from MDCK cells (A, lane 1) was resolved on a 6% SDS polyacrylamide gel, transferred to nitrocellulose, and processed for immunoblotting using rabbit antibodies specific for ank3. Ank3 antibodies were also used to prepare immunoprecipitates from MDCK cells labeled with ³²Porthophosphate at 37°C for 4 hours in the absence (A, lanes 2 and 4) or presence (A, lanes 3 and 5) of 120 µm TBB. In addition, immunoprecipitates prepared from unlabeled MDCK cells were incubated in kinase buffer containing [-³²P]-ATP for 30 minutes at 37°C (B). In some instances, 50 µg of heparin was included during the in vitro kinase reaction (B, lane 3). Alternatively, the precipitate was boiled for 10 minutes, which eliminated the activity of the coprecipitating kinase (B, lane 4), and subsequently incubated with 15 mU of purified CK2 from rat liver (B, lane 5). Samples were analyzed on 6% SDS polyacrylamide gels. Immunoprecipitated polypeptides were visualized by staining with GelCode Blue, and ³²P-labeled species were detected by autoradiography. The migration of the 215 kDa and 200 kDa ank3 isoforms is indicated to the left of each panel.

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