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. 2002 Mar;22(5):1288–1297. doi: 10.1128/mcb.22.5.1288-1297.2002

FIG. 3.

FIG. 3.

Kin28 is phosphorylated on T162 within the T loop. (A) Immunoblot analysis of wild-type (WT) Kin28 whole-cell extracts (WCE) shows that the protein migrates as a doublet corresponding to the phosphorylated and unphosphorylated species. The T162A mutant protein migrates as a single band. Kin28*p, phosphorylated Kin28; TBP, TATA-binding protein. (B) HA-tagged Kin28 was immunoprecipitated (IP) via the HA tag using the 12CA5 monoclonal antibody and treated with λ-phosphatase (Ppase). The faster-migrating band disappears, indicating that the mobility shift is phosphorylation dependent. The HA-tagged Kin28(T162A) [Kin28.HA(T162A)] mutant was used as a gel mobility standard for the unphosphorylated form.