Figure 2. Characterization of Net1 phosphorylation on S131 and T146 in cells.

(A) Characterization of pS131- and pT146-specific antibodies. HeLa cells were transfected with HA-epitope tagged wild type or mutant Net1 expression vectors and synchronized in prometaphase by overnight treatment with nocodazole. Mitotic cells were isolated by shakeoff, lysed, and HA-tagged proteins were immunoprecipitated. Immunoprecipitated proteins were resolved by SDS-PAGE and examined by western blotting. Shown is a representative experiment from 6 independent experiments. (B) Phosphorylation of S131 and T146 in synchronized cells. HeLa cells were transfected with HA-tagged Net1 and synchronized in G₀ by serum starvation, S phase by double thymidine treatment, of pro-metaphase by nocodazole treatment. HA-Net1 was then immunoprecipitated and analyzed by western blotting. Shown is a representative experiment from 4 independent experiments. (C) pS131 and pT146 is sensitive to Cdk inhibition. HeLa cells were transfected with HA-Net1, synchronized in pro-metaphase, and treated with vehicle or the Cdk inhibitor roscovitin (10 μM) for 2 hours. HA-Net1 was then immunoprecipitated and analyzed by Western blotting. Phosphorylation of Lamin A/C on the Cdk1 site S22 and Cdk1 on the activation loop site T161 was assessed in the lysate to confirm inhibitor function. Shown is a representative experiment from 3 independent experiments. (D) Assessment of specificity of pS131 and pT146 antibodies for endogenous Net1. HeLa cells transfected with control or Net1-specific siRNAs and then synchronized in prometaphase. Cells were lysed and tested for pS131 and pT146 by western blotting. Shown is a representative experiment from 3 independent experiments. (E) Phosphorylation of endogenous Net1 on S131 and T146 is enriched in mitotic cells. HeLa cells were grown as an asynchronous population or synchronized in pro-metaphase. Cells were then lysed in an SDS-containing buffer and tested for phosphorylation of endogenous Net1 on S131 and T146 by western blotting. Shown is a representative experiment from 3 independent experiments. (F) Endogenous Net1 phosphorylation on S131 and T146 is sensitive to Cdk inhibition. HeLa cells were synchronized in pro-metaphase and treated with vehicle or roscovitin (10 μM) for 2 hours. Phosphorylation of Net1 was assessed by western blotting. Shown is a representative experiment from 4 independent experiments.